Novel polyketides and antibiotics

ABSTRACT

Facile methods for preparing diketide and triketide thioesters are disclosed. The resulting thioesters may be used as intermediates in the synthesis of desired polyketides, and may contain functional groups which ultimately reside in side chains on the resulting polyketide and thus can be used further to manipulate the polyketide so as form derivatives. The polyketides produced may also be tailored by glycosylation, hydroxylation and the like. New polyketides and their derivatives and tailored forms are thereby produced.

[0001] This application claims priority under 35 U.S.C. § 119 from U.S.Ser. No. 60/117,384 filed Jan. 27, 1999. The contents of thisapplication are incorporated herein by reference.

TECHNICAL FIELD

[0002] The invention concerns methods for the efficient synthesis ofoligoketide thioesters, including diketide and triketides, which areuseful as intermediates in polyketide production and to methods to usethese intermediates. The methods of synthesis are suitable for liquidphase as well as solid-phase combinatorial synthesis. The invention alsoincludes polyketide and tailored polyketide products.

BACKGROUND OF THE INVENTION

[0003] The creation of novel macrolide polyketides has been achievedthrough genetic manipulation of polyketide synthases. The modular natureof the Type1polyketide synthases allows for domain exchange betweendifferent polyketide synthase genes, resulting in hybrid genes whichproduce polyketide synthases with altered properties that result, inturn, in modified macrolide structures. Thus, it is possible to controlchain length, choice of chain extender unit, degree of β-carbonoxidation level, and to some degree stereochemistry. The choice ofstarter unit has been more difficult to control. Two complementaryapproaches have been described.

[0004] Dutton, et al., J. Antibiotics (1991) 44:357-365 demonstratedthat the avermectin polyketide synthase was somewhat flexible in choiceof starter units. When denied the natural starter unit throughinactivation of the branched-chain amino acid dehydrogenase, theavermectin polyketide synthase will accept a variety of α-branchedcarboxylic acids as the starter unit. However, only about 30 acids outof nearly 800 candidate acids tried were accepted. Acids without anα-branch appear to be metabolized through β-oxidation until an α-branchis reached, further limiting this methodology. Marsden, et al., Science(1998) 79:199-202 exchanged the native loading domain of theerythromycin PKS with that from the avermectin polyketide synthase,resulting in a hybrid PKS having the same loosened starter unitspecificity as the avermectin PKS. Clearly, the native specificities ofenzymatic domains will always be a limitation on the flexibility ofresulting hybrid systems.

[0005] A more general method for controlling starter unit specificityhas been described by Jacobsen, et al., Science (1997) 277:367-369.Inactivation of the ketosynthase in module 1 (KS1) of the erythromycinPKS (DEBS) results in an enzyme (KS1°-DEBS) incapable of initiatingpolyketide synthesis using precursors normally available to the cell.When supplied with a suitable thioester of the diketide product ofmodule 1 or its analogs, however, KS1°-DEBS efficiently incorporatesthese into full-length polyketides. Subsequent experiments havedemonstrated that a very wide range of diketide analogs are accepted byKS1°-DEBS, making this a very general method for production of analogsof the polyketide precursor of erythromycin, 6-deoxyerythronolide B(6-dEB), with variations at the positions controlled by the starterunit. Further, this method allows for production of 6-dEB analogsaltered at the 12-position; this is equivalent to altering the substratespecificity of the module 1 acyltransferase (AT1) which transfers thefirst extender unit. While this has been accomplished through the abovedescribed domain exchange experiments as well, the “diketide method”allows for introduction of 12-position substituents which are notavailable from nature. Furthermore, triketide analogs are accepted,opening the 10- and 11-positions of 6-dEB for modification. The 6-dEBanalogs obtained can be further converted into analogs of erythromycinby feeding to a suitable converter strain, such as a strain ofSaccharopolyspora erythraea containing a non-functional erythromycinPKS. The resulting erythromycins have altered side-chains at the13-position as well as other optional modifications, and show alteredbiological activity. These erythromycin analogs can also be produced byintroducing the KS1°-mutation into an erythromycin-producing strain ofSaccharopolyspora erythraea, then supplying the mutant strain withsuitable diketide or triketide thioesters as described above.

[0006] Implementation of this method requires the availability of theN-acylcysteamine oligoketide thioesters. Synthetic methods available inthe art for these thioesters do not lend themselves to efficient,economical synthesis, or to the systematic production of variants. Thediketide and triketides also typically contain chiral centers requiringthe control of absolute or relative stereochemistry.

[0007] Cane, D. E., et al., J Am Chem Soc (1987) 109:1255-1257 describesa three-step process to produce the N-acetylcysteamine thioester of(2S,3R)-2-methyl-3-hydroxy pentanoic acid. The method relies on the useof a chiral reagent N-propionyl-(4S)-4-isopropyl-2-oxazolidinone forcontrol of the absolute stereochemistry of the product:

[0008] This material results from the acylation of(4S)-4-isopropyl-2-oxazolidinone with propionyl chloride, typicallyusing a strong base such as n-butyllithium at low temperature. The Caneprocess is an aldol condensation of this starting material withpropionaldehyde in the presence of dibutylboron triflate (Bu₂BOTf),followed by hydrolysis of the imide (lithium hydroperoxide) andthioesterification with N-acetylcysteamine in the presence of diphenylphosphorylazide and triethylamine. This multi-step process isinefficient, with substantial losses accompanying the hydrolysis step.

[0009] Cane, D. E., et al., J Antibiotics (1995) 48:647-651 was able toimprove yields using a five-step process which replaces the aldolcondensation with a Claisen condensation between the lithium enolate ofthe propionyl oxazolidinone (N-propionyl-(4S)-4-benzyl-2-oxazolidinonewas used as the stereochemistry controlling starting material in thismethod) and propionyl chloride followed by reduction of the resultingβ-ketoester product using zinc borohydride. Protection of the β-hydroxysubstituent as a tert-butyldimethylsilyl ether preceded hydrolysis ofthe imide, which again required lithium hydroperoxide. The protectinggroup gave improved yields from hydrolysis, but required an additionaltwo steps to add and remove. This longer process also suffers from theuse of zinc borohydride, which is not commercially available.

[0010] Cleavage of the N-acyloxazolidinones resulting from either aldolor Claisen condensations as described above is problematic. Variousmethods of cleavage are known in the art, including that of Evans, D.A., et al., Tetrahedron Lett (1987) 28:6141, in which undesired reactionat the oxazolidinone carbonyl during hydrolysis is suppressed by the useof lithium hydroperoxide. This process requires the use of concentratedsolutions of hydrogen peroxide, which are explosive and dangerous forlarge-scale processes. The N-acyloxazolidinones are unreactive towardsthiols or thiolates, although some conversion to thioesters can beobserved using concentrated solutions of lithium thiolates intetrahydrofuran. The low solubility of the thiolates in tetrahydrofurancombined with epimerization of chiral diketides due to the basicity ofthe thiolates limits the utility of this method. Miyata, O., et al., SynLett (1994) 637-638, describes conversion of N-acyloxazolidinones toS-benzylthioesters through the use of lithiumbenzylthiotrimethylaluminate. The production of more complex thioesterscontaining groups capable of binding Lewis acids like trimethylaluminum,such as those based on N-acylcysteamine, has not been reported.

[0011] The N-acetylcysteamine thioesters of larger oligoketides havealso been prepared. Cane, D. E., et al., J Am Chem Soc (1993)115:522-526 synthesized the N-acetylcysteamine thioester of(4S,5R)-5-hydroxy-4-methyl-2-heptenoic acid using the stereochemicallycontrolled aldol condensation product ofN-propionyl-(4S)-4-benzyl-2-oxazolidinone as the starting material:

[0012] This imide was converted to the corresponding aldehyde, andextended at the carbonyl group by a Wittig reaction to obtain thedesired triketide as the ethyl ester which was then hydrolyzed andconverted to the acylcysteamine thioester in a two-step process. Yieldswere improved by addition of steps protecting the alcohol, Cane, D. E.,et al, J Am Chem Soc (1993) 115:527-535. However, this approach clearlydoes not lend itself to efficient modular solid-phase synthesis sincethe building of the triketide chain is nonlinear—i.e., the condensationsand the Wittig reactions extend the diketide in opposing directions.Each new analog requires complete passage through the synthesis with nocommon intermediates.

[0013] While it is clear that thioester forms of acyl moieties,diketides and triketides can be incorporated by PKS systems, to date,little has been reported concerning the optimal thioesters to producethe desired polyketides other than that N-acetylcysteamine thioestersare generally effective as compared with the free carboxylic acids ortheir oxy-esters (Cane & Yan). It may be expected, however, that thenature of the thioester, e.g. the acyl group in an N-acylcysteaminethioester, might influence such important factors as water solubility,transport into the bacterial cell, metabolism, and recognition by thePKS. A synthetic method for producing variation in the thioester groupitself would thus be advantageous.

[0014] The present invention offers both improved efficiency in thesynthesis of optically-pure diketide thioester intermediates and anapproach which provides for efficient extension of the diketides intothe corresponding triketide thioesters and provides for additionalcondensation steps to extend the oligoketide still further. The presentinvention further provides a method for synthesis of racemic rather thanoptically pure diketide thioesters. The racemic materials constitutelow-cost alternatives for large-scale production of novel polyketides byfermentation.

DISCLOSURE OF THE INVENTION

[0015] The invention offers improvements in the synthesis of oligoketidethioester intermediates. These intermediates can then be incorporatedinto pathways for the synthesis of novel polyketides using native ormodified polyketide synthase (PKS) systems. The invention offers animprovement in the efficiency of diketide synthesis as well as a methodfor synthesis of triketides and oligoketides in general which is adaptedto efficient, linear, solid-phase synthesis. The invention furtherprovides a method to produce racemic diketide thioesters in aneconomical manner. The invention further provides a method for producingnovel polyketides suitable for further modification through theintroduction of unique functionalities.

[0016] Thus, in one aspect, the invention is directed to the conversionof an acyl imide such as that of a diketide, triketide or oligoketidedirectly to an N-acylcysteamine thioester by treating the imide with asalt of the corresponding mercaptan. For the synthesis of opticallyactive oligoketide thioesters starting from chiral oxazolidinones, thisis done in the presence of a Lewis acid to facilitate the reaction andpreserve stereochemical purity. For the synthesis of racemic oligoketidethioesters starting from achiral benzoxazolones, the Lewis acid is notrequired. This method obviates the intermediate steps of imidehydrolysis, alcohol protection, thioesterification, and deprotectionused by previous methods. This method is particularly advantageous forsolid-phase synthesis, as it allows for generation of the product withsimultaneous cleavage of the oligoketide from the solid support. Aparticularly facile process using transthiolation of thioesters isgiven.

[0017] In a second aspect, the invention is directed to a method tosynthesize racemic diketides and their derivatives through thetitanium-mediated aldol condensation between N-acyl-2-benzoxazoloneswith aldehydes, followed by reaction of the aldol products withnucleophiles to yield the desired derivatives. This method provides adirect route to various oligoketide derivatives, including esters andamides, and is particularly advantageous for the multi-kilogram,economical synthesis of diketide N-acylcysteamine thioesters requiredfor fermentation. As the relative chirality of the carbons at positions2 and 3 of the attached acyl group is preserved, the racemic mixturewill contain one isomer which can be utilized by the PKS and only oneadditional isomer which cannot. This is in contrast to production of thefour possible diestereomers which would result in utilization of onlyone-quarter of the available molecules.

[0018] Thus, in still another aspect, the invention is directed tomethods to synthesize diketides and triketides which can be used toproduce macrolides with functional substituents for example at the 13-and 14-positions by employing, for example, alkenyl- orbenzyloxy-aldehydes to introduce starter unit and/or first extendermoiety equivalents containing derivatizable groups. The benzyloxy groupcan readily be converted to a hydroxyl by reduction and then mesylatedto provide a suitable leaving group for replacement with nucleophiles,including halides, azides, amines, thiols, other alcohols, and cyanide.The alkenyl group can be functionalized by any of numerous methods knownin the art, including Heck coupling to introduce aryl groups. Suchderivatizations can be performed either on the oligoketide or on thepolyketides which are produced upon feeding of the oligoketides tosuitable PKS systems or cultures of microorganisms.

[0019] In an additional aspect, the invention is directed to methods tosynthesize oligoketide thioesters using solid-phase combinatorialchemistry. These methods are particularly advantageous when a library ofoligoketide thioesters is desired.

[0020] In summary, because the invention permits a wide variety ofdiketide and triketide thioesters to be synthesized in a facile andeconomic-manner, it is possible to prepare a wide variety of polyketidesand their tailored derivatives taking advantage of the availability ofboth recombinant and natively produced polyketide synthase systems andtailoring enzymes, as well as employing chemical transformations usingside-chain functional groups.

[0021] In still other aspects, the invention relates to feedingdiketides or triketides, prepared by the methods of the invention, tosuitable PKS systems in vitro or in vivo to obtain oligoketides orpolyketides and further converting said polyketides to antibiotics byglycosylation and/or other modifications. The invention also relates tonovel intermediates and the resulting modified polyketides andantibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 sets forth structures of illustrative suitable N-acylcysteamines.

[0023]FIG. 2 illustrates the method for conversion ofN-acyloxazolidinones into N-acylcysteamine thioesters.

[0024]FIG. 3 illustrates methods for the conversion ofN-acyl-2-benzoxazolones into various acyl derivatives, includingN-acylcysteamine thioesters.

[0025]FIG. 4 illustrates the transthioesterification method developedfor use with the diketide benzoxazolones.

[0026]FIG. 5 illustrates the formation of N-acyl-2-benzoxazolone and thealdol condensation between N-acyl-2-benzoxazolones and aldehydes, usedto prepare intermediates for the synthesis of racemic diketides.

[0027]FIG. 6 illustrates the rationale for enforcement ofsyn-stereochemistry by the benzoxazolone auxiliary.

[0028]FIGS. 7A and 7B illustrate typical diketides, shown as theirN-acylcysteamine (SNAC) thioesters, prepared according to the invention.

[0029]FIG. 8 illustrates synthesis of oligoketide thioesters usingsolid-phase chemistry.

MODES OF CARRYING OUT THE INVENTION

[0030] The invention provides methods useful in the synthesis ofintermediates for the production of polyketides that havecharacteristics desirable for efficient and practical applications.

[0031] An efficient synthetic strategy for the required oligoketidethioesters should provide:

[0032] 1) stereochemical control;

[0033] 2) a minimum number of synthetic steps;

[0034] 3) synthetic steps with high yields;

[0035] 4) use of common intermediates;

[0036] 5) adaptability to solid phase synthesis;

[0037] 6) adaptability to combinatorial library generation.

[0038] For large-scale applications, further criteria concern the costand availability of reagents.

[0039] The invention methods provide these characteristics. The crucialstep in all routes to diketide thioesters is the formation of thethioester linkage. Due to low reactivity of the commonly usedN-acyloxazolidinone intermediates towards thiol nucleophiles, thisprocess usually requires several steps as described above. Theoxazolidinone auxiliary is removed by hydrolysis, and the resulting acidis activated and converted into the thioester. It is possible to convertN-acyloxazolidinones into thioesters directly by treatment with thelithium salt of the mercaptan, but yields are typically low and loss ofstereochemical integrity is often noted for chiral diketides. Theinvention provides an efficient method for direct conversion ofN-acyloxazolidinones into thioesters of N-acylcysteamines which uses thetrimethylaluminum complex of the lithium mercaptide (FIG. 2). Thisproceeds in high yields without detectable loss of stereochemicalintegrity. Since the filing of provisional application 60/117,384, asimilar method using trimethylaluminum with N-acetylcysteamine has beenreported in C. LeSann, et al., Tetrahedron Letters (1999) 40:4093-4096.

[0040] The invention also provides a method for the direct conversion ofN-acyl-2-benzoxazolones into N-acylcysteamine thioesters by simpletreatment with an alkali metal salt of the mercaptan in an alcoholsolvent (FIG. 3). This is efficient and mild due to the more readydisplaceability of the benzoxazolone as compared with an oxazolidinoneand the lower basicity of thiolates in protic solvents. Methods for theconversion of the N-acylbenzoxazolones into other functional groups arealso illustrated. The alkali metal salt of the mercaptan may begenerated through the reaction of a mercaptan with a metal alkoxide,such as sodium methoxide or sodium ethoxide, under inert atmosphere soas to prevent disulfide formation. In a particularly simple embodiment,the required alkali metal salt of the mercaptan is generated in situthrough treatment of a alcoholic solution of a simple thioester, e.g.,N,S-diacylcysteamine, with one molar equivalent of an alkali metalalkoxide. Addition of the N-acylbenzoxazolone where acyl is anoligoketide then provides the oligoketide thioester. Suitable alcoholsare methanol, ethanol, isopropanol, and related solvents. Suitablealkali metal alkoxides are those derived from the aforementioned alcoholsolvents, such as lithium methoxide, sodium methoxide, potassiummethoxide, and similar salts of the other alcohols. The reaction istypically performed at ambient temperatures. This method has theadvantage of avoiding disulfides which are typically present in freemercaptans due to air oxidation. The N,S-diacylcysteamines are readilyavailable through the reaction of cysteamine hydrochloride with anexcess of the acyl anhydride in water in the presence of a suitablebase. A convenient base is saturated aqueous sodium bicarbonate, whichprovides a pH where the thioester product is stable. UnlikeN-acylcysteamines, the N,S-diacylcysteamines are typically crystalline,stable materials which can be stored indefinitely.

[0041] The invention further provides a method for synthesis of racemicdiketides using 2-benzoxazolone as supporting auxiliary (FIG. 4). Thetitanium tetrachloride-mediated aldol condensation betweenN-propionyl-2-benzoxazolone and an aldehyde provides high yields ofthese diketides, with excellent diastereochemical control.“Benzoxazolone derivative” means, generally, the imide of an acyl groupwith a 2-benzoxazolone. The aromatic moiety in benzoxazolone may beunsubstituted as in benzoxazolone per se or may be substituted, forexample as is the case for chlorzoxazone. Alternative substitutions onthe benzylidene moiety may also be employed, such as bromo, methyl, andthe like. Both 2-benzoxazolone and 5-chloro-2-benzoxazolone(chlorzoxazone) have been shown to be effective auxiliaries,supporting >95% syn aldol condensation for simple aldehydes and ca. 90%syn aldol condensation with sterically-hindered aldehydes such aspivalaldehyde and with chelating aldehydes such as α-alkoxyaldehydes.The titanium aldol condensation has further advantages in that it can beperformed at moderate temperatures (0° C.), unlike reactions of lithiumenolates which require the use of −78° C., and in that the reagents areextremely inexpensive ($10/mol) as compared with dibutylboron triflate($750/mol). Further, the oxidative workup using concentrated hydrogenperoxide needed with boron-mediated aldols is not required.

[0042] The use of N-propionyl-2-benzoxazolone in the aldol condensationprovides diketides of benzoxazolones having a 2-methyl substituent,which, in turn, provides a 12-methyl group in the 6-dEB analog obtainedupon conversion of the diketide by the erythromycin PKS. Similarly, theuse of N-crotonyl-2-benzoxazolone ultimately provides diketides having a2-vinyl substituent, which provides a 12-vinyl group in the 6-dEB analogobtained upon conversion of the diketide by the erythromycin PKS. OtherN-acyl-2-benzoxazolones can be used to provide other 2-substituteddiketides, and thus other 12-substituted 6-dEB and erythromycin analogs.

[0043] The invention further provides a method for introducingsubstituents at the 12-, 14-, and 15-positions of 6-dEB or erythromycinwhich are not tolerated by the erythromycin PKS and thus cannot beintroduced directly by feeding the corresponding oligoketide thioester.This method involves feeding an oligoketide thioester containing afunctional group, typically an alkene or a protected alcohol group,which is tolerated by the PKS and which can be converted post-PKS intothe desired functionality using chemical, enzymic, or biologicalconversion. For example, the erythromycin PKS will efficiently convertdiketides containing alkene groups either at the 2- or 3-positions (orboth) to provide the corresponding 12- or 13-vinyl 6-dEB analogs. Theerythromycin PKS will convert 3-hydroxy-2-methyl-4-pentenoateN-acylcysteamine thioesters into 14,15-dehydro-6-dEB, for example, andthe post-PKS enzymes of Saccharopolyspora erythraea will convert thisfurther into 14,15-dehydroerythromycins. This introduces a unique alkenefunctionality into the 6-dEB and erythromycin molecules. Methods forconversion of this alkene, e.g., into halides, carbonyls, alcohols,ethers, and amines are well known in the art. The alkene can also beused to add aromatic moieties onto the 6-dEB or erythromycin moleculethrough the Heck reaction:

[0044] Similarly, the erythromycin PKS will convert3-hydroxy-2-vinylpentanoate N-acylcysteamine thioesters into12-desmethyl-12-vinyl-6-dEB. This provides a unique alkene functionalityat the 12-position of 6-dEB which can be further manipulated. As anextension of this concept, Streptomyces coelicolor CH999 expressing theplasmid p3RJ2 converts 3-hydroxy-2-vinyl-6-heptenoate N-acylcysteaminethioesters into 12,15-divinyl-12-desmethyl-6-dEB at levels ofapproximately 50 mg/L:

[0045] This compound can be subsequently converted into variousderivatives, such as the bicyclic analog illustrated through the use ofan olefin metathesis catalyst.

[0046] Other protected or masked functionalities can be introduced intothe 6-dEB and erythromycin molecules in this fashion. For example,alcohols protected as esters or as benzyl ethers would be suitableprecursors which would allow for introduction of a new alcohol group inthe polyketide. The modification of alcohols into other functionalgroups is well known in the art.

[0047] This methodology can also be used to introduce reactivefunctionalities directly. As an example, the erythromycin PKS willconvert 5-halo-3-hydroxy-2-methylpentanoate N-acylcysteamine thioestersinto the corresponding 15-halo-6-dEBs. The halogen can be F, Cl, Br, orI, and supplies a readily-displaceable group for subsequent modificationof the 15-position of the 6-dEB or erythromycin.

[0048] It can be seen, therefore, that the feeding of synthetic diketidethioester analogs to the erythromycin PKS or an organism expressing theerythromycin PKS is a useful means of producing novel polyketides. Themethod is particularly useful when the PKS has been modified so as topreclude formation of the natural polyketide, such as by inactivation ofthe module 1 ketosynthase, or more generally when the supply of naturalstarter unit has been otherwise eliminated.

[0049] It can be seen as well that methods existing in the art forconstruction of lengthier oligoketides can be adapted for use in thesesystems. For example, triketides are readily available by aldolcondensations between aldehydes and β-ketoimides as described in D. A.Evans, et al., J. Am. Chem. Soc. (1990) 112:866-868. Methods have beendeveloped for the efficient control of relative stereochemistry in thesetransformations, as described in D. A. Evans, et al., Tetrahedron (1992)48:2127-2142. The stereoselective reduction of the resulting productsusing triacetoxyborohydrides has been described in D. A. Evans, et al.,J. Am. Chem. Soc. (1988) 110:3560-3578, and provides a means of furtheraltering the functionality of the oligoketides by selective introductionof a β-hydroxyl. Such hydroxyls can be further converted into alkenesthrough acylation and β-elimination, with the proviso that otherhydroxyls in the oligoketide must be protected against acylation or atleast must be readily deprotected afterwards. The invention provides aparticularly simple method for this transformation using phosgene toform a cyclic carbonate, which simultaneously activates the β-hydroxylfor elimination and protects the δ-hydroxyl:

[0050] Construction of triketides:

[0051] Alternatively, the alkene resulting from elimination can bereduced to the alkane, for example by catalytic hydrogenation, prior tothiolysis. Thus, all four reductive outcomes observed from naturalpolyketide synthesis can be mimicked in the chemical construction oftriketides.

[0052] The direct, efficient conversion of oligoketide imides intothioesters opens the possibility of efficient solid-phase synthesis ofoligoketide thioesters, as thiolysis can be used to free the oligoketidechain from the solid support as the final step in the synthesis. Methodsexist for the linear elaboration of oligoketides wherein the oligoketidechain is grown off an initial auxiliary unit, typically a chiraloxazolidinone, at least up to the triketide level. The number ofrequired steps is minimal and the yields are high. The use of the4-benzyl-2-oxazolidinone residue maintains the stereochemistry throughmultiple chain extensions using similar reagents. Because commonintermediates are used and the auxiliary stereochemistry controllingcompound can readily be linked to solid supports as described in theinvention, the method provides a suitable basis for the solid-phaseproduction of combinatorial libraries of triketides and beyond.

[0053] Two possible attachment sites to a solid support can beenvisioned. By providing functionality on the phenyl group of the4-benzyloxazolidinone, covalent coupling to a wide variety of supportsmay conveniently be obtained; means for coupling through this moiety arewell known in the art. For example, the corresponding auxiliary derivedfrom tyrosine rather than phenylalanine can be readily prepared. Thiswould provide a phenolic hydroxyl group which could readily be attachedto a solid support through, for example, reaction with a chlorobenzylpolystyrene resin to give a diphenylether-linked chiral oxazolidinone.

[0054] The oligoketide chain can be grown on this support using methodswell-established in the art, then cleaved from the solid support,preferably by formation of the thioester as described herein. Thisoffers an advantage over previous methods for solid-phase oligoketidesynthesis, e.g., Reggelin, M., et al., Tetrahedron Letters (1996)37:6851-6852, in which the oligoketide chain itself is used as theattachment point, with a corresponding attachment functionalityremaining as part of the oligoketide at the end of the synthesis.

[0055] Alternatively, the oxazolidinone ring itself is used as the pointof attachment to the support. For example, the solid support of theinvention may employ a chiral imidazolidinone, wherein theimidazolidinone replaces the oxazolidinone described above:

[0056] The second nitrogen atom of the imidazolidinone is used as theattachment point to the solid support, leaving the nitrogen adjacent tothe chiral center (equivalent to the nitrogen in an oxazolidinone) openfor acylation with an acyl chloride. The use of untethered chiralimidazolidinones as synthetic auxiliaries has been described by S. E.Drewes, et al., Chem. Ber. (1993) 126:2663, and an especially facilemethod for their acylation has been described by W. M. Clark & C.Bender, J. Org. Chem. (1998) 63:6732.

[0057] The tethered chiral imidazolidinones can be readily prepared fromoptically pure amino acids by standard procedures; e.g., by conversionof a chiral amino acid such as phenylalanine into the carbamate byreaction with a suitable chloroformate or chloroformate equivalent,followed by conversion to the aminoaldehyde (Rittel, K. E., et al., JOrg Chem (1988) 47:3016; Organic Syntheses, vol. 67:69-75) andsubsequent reductive amination to add a suitable functionalized linker.

[0058] The functional group, shown as “X” is, for example, an amine, acarboxylate or an ester, thiol or halide which is used to attach theauxiliary to a solid support. The resulting amino carbamate is cyclizedto the imidazolidinone by treatment with a suitable base or with heat.

[0059] Racemic diketides can be synthesized on solid supports using asimilar technique. In this case, the 2-benzoxazolone auxiliary can beattached to the support by either of two methods. Halogenatedbenzoxazolones, such as chlorzoxazone (5-chloro-2-benzoxazolone), arereadily available and provide a simple means of attachment through thearomatic halide. For example, chlorzoxazone can be coupled with analkene-containing support using palladium catalysis (the Heck reaction).Alternately, 2-benzimidazolone (2-hydroxybenzimidazole) can be coupledto a support through one of the imidazolone nitrogens, leaving thesecond free for acylation as described above.

[0060] The methods described above for elaboration of triketides areideally suited to solid-phase synthesis, as the directing auxiliarygroup (oxazolidinone or benzoxazolone) remain attached to the growingoligoketide chain. The attached auxiliary then serves as a leaving groupduring thioester formation, yielding an oligoketide thioester with noresidue remaining from the solid support.

[0061] Incorporation into Polyketides

[0062] As used herein, “polyketide” refers to the immediate product of apolyketide synthase enzyme system. It is generally a lactone of 13-15C.An example of a polyketide would be 6-dEB, the immediate product of theerythromycin PKS. “Tailored polyketides” refers to the products ofsubsequent derivatization of the resultant polyketide which occursthrough enzymatic treatment by enzymes endogenous to organisms whichsynthesize polyketide antibiotics. Such tailoring enzymes may addhydroxyl groups, remove hydroxyl or oxo groups, add sugars, modifysugars that have been coupled to the polyketide, and the like.“Derivatized polyketides” refers to polyketides or tailored polyketideswhich have been modified chemically in ways generally unavailable fromstraightforward enzymatic treatment. The diketides and triketidesprepared by the methods of the invention, because they containfunctional groups which can further be reacted result in polyketides andtailored polyketides that can be derivatized using synthetic chemicalreactions. Methods for further converting polyketides (or tailoredpolyketides) are found, for example, in PCT publications WO 99/35156 andWO 99/35157, incorporated herein by reference. Such methods are alsodescribed in U.S. Ser. Nos., respectively, 60/172,154 and 60/172,159,both filed Dec. 17, 1999; 60/173,805 filed Dec. 30, 1999; and 60/173,804filed Dec. 30, 1999, and each incorporated herein by reference.

[0063] The thioesters of the diketides and triketides of the inventioncan be incorporated into polyketides by the PKS system, mostadvantageously when competition from the native starter unit iseliminated by, for example, the inactivation of the ketosynthase domainin module 1 as described in PCT application PCT/US96/11317 incorporatedherein by reference. Polyketide synthases thus modified are alsodescribed in U.S. Ser. No. 08/896,323 filed Jul. 17, 1997 andincorporated herein by reference. As described in these applications,the polyketide synthase system can be employed in a cell-free context,or can be utilized in vivo either in its native host or in a recombinanthost cell. For example, the organism which natively produceserythromycin, Saccharopolyspora erythreae may be used, or, as set forthin U.S. Pat. No. 5,672,491, the entire erythromycin gene cluster can beinserted into a suitable host such as Streptomyces coelicolor or S.lividans, preferably a S. coelicolor or S. lividans which has beenmodified to delete its endogenous actinorhodin polyketide synthesismechanism. A typical host would be S. coelicolor CH999/pJRJ2, whichexpresses a mutant 6-deoxyerythronolide B synthase having an inactivatedmodule 1 ketosynthase (J. Jacobsen, et al., 1997 Science 277:367-369).The diketides or triketides are thus incorporated into the resultingpolyketide. In the case of the diketides and triketides provided by thisinvention, the resulting erythronolide will be correspondingly modifiedat positions 10-15. For example, feeding a growing culture of S.coelicolor CH999/pJRJ2 with(2S,3R)-5-fluoro-3-hydroxy-2-methylpentanoate N-acetylcysteaminethioester results in production of 15-fluoro-6-deoxyerythronolide B,while feeding with (2S,3R)-3-hydroxy-2-methylhexanoateN-acetylcysteamine thioester results in production of15-methyl-6-deoxyerythronolide B. Feeding S. coelicolor CH999/pJRJ2 with(2S,3R)-3-hydroxy-2-vinylpentanoate N-acetylcysteamine thioester resultsin production of 12-desmethyl-12-vinyl-6-deoxyerythronolide B.

[0064] Further, the diketide or triketide intermediates can be providedto PKS enzymes other than the 6-dEB synthase of Saccharopolysporaerythraea. Other PKS enzymes include the 6-dEB synthase ofMicromonospora megalomicea and its KS1° derivative described in U.S.Ser. No. 60/158,305, filed Oct. 8, 1999; the oleandolide PKS and itsKS1° derivative described in PCT application No. US99/24478, filed Oct.22, 1999; and the narbonolide PKS and its KS1° derivative described inPCT publication No. WO 99/61599, published Dec. 2, 1999, allincorporated by reference.

[0065] The diketides and triketides can be provided to a host cell thatexpresses a PKS but not post PKS modification enzymes (such ashydroxylases and glycosyltransferases) or can be provided to a host cellthat expresses both types of enzymes.

[0066] Recombinant host cells containing cloned PKS expression vectorscan be constructed to express all of the biosynthetic genes for amodified polyketide or only a subset of the same. If only the genes forthe PKS are expressed in a host cell that otherwise does not producepolyketide modifying enzymes that can act on the polyketide produced,then the host cell produces unmodified polyketides. Such unmodifiedpolyketides can be hydroxylated and glycosylated, for example, by addingthem to the fermentation of a strain such as, for example, Streptomycesantibioticus or Saccharopolyspora erythraea, that contains the requisitemodification enzymes.

[0067] If desired, further modifications at positions 14 and 15 areachievable once the resulting polyketide is isolated by employing anappropriate benzyloxy-, alkene-, or halo-substituted diketide thioester.As set forth above, these groups can be converted to otherfunctionalities using methods well known in the art.

[0068] The resulting polyketides can further be modified by chemicalmeans or by feeding to a native antibiotic producing host forglycosylation or further modification. For example, a resulting6-deoxyerythronolide can be fed to Sac. erythraea for hydroxylation atthe 6- and/or 12-positions and sugar attachment at the 3- and/or5-positions. This is particularly useful when the organism used containsa defective PKS gene, resulting either from random mutagenesis or fromdesigned deletion. The strain Sac. erythraea K39-14 expresses adefective 6-deoxyerythronolide B synthase, and so is incapable ofproducing erythromycins under normal fermentation conditions. Feeding agrowing culture of Sac. erythraea K39-14 with15-fluoro-6-deoxyerythonolide B results in production of15-fluoroerythromycins. Feeding this strain with15-methyl-6-deoxyerythronolide B results in formation of15-methylerythromycins. Both 15-fluoroerythromycin A and15-methylerythromycin A have been found to have strong antibacterialactivity.

[0069] In lieu of, or in addition to chemical synthesis steps, theinitially produced polyketides can be “tailored.” There is a widevariety of diverse organisms that can modify polyketides and/or theirderivatives to provide compounds with, or that can be readily modifiedto have, useful activities. As stated above, Saccharopolyspora erythraeacan convert 6-dEB to a variety of useful compounds. The erythronolide6-dEB is converted by the eryF gene product to erythronolide B, whichis, in turn, glycosylated by the eryB gene product to obtain3-O-mycarosylerythronolide B, which contains L-mycarose at C-3. Theenzyme eryC gene product then converts this compound to erythromycin Dby glycosylation with D-desosamine at C-5. Erythromycin D, therefore,differs from 6-dEB through glycosylation and by the addition of ahydroxyl group at C-6. Erythromycin D can be converted to erythromycin Bin a reaction catalyzed by the eryG gene product by methylating theL-mycarose residue at C-3. Erythromycin D is converted to erythromycin Cby the addition of a hydroxyl group at C-12 in a reaction catalyzed bythe eryK gene product. Erythromycin A is obtained from erythromycin C bymethylation of the mycarose residue in a reaction catalyzed by the eryGgene product. The unmodified polyketide compounds provided by thepresent invention can be provided to cultures of S. erythraea andconverted to the corresponding derivatives of erythromycins A, B, C, andD in accordance with the invention. To ensure that only the desiredcompound is produced, one can use an S. erythraea eryA mutant that isunable to produce 6-dEB but can still carry out the desired conversions(Weber, et al., 1985, J. Bacteriol. 164(1):425-433). Also, one canemploy other mutant strains, such as eryB, eryC, eryG, and/or eryKmutants, or mutant strains having mutations in multiple genes, toaccumulate a preferred compound. The conversion can also be carried outin large fermentors for commercial production.

[0070] There are other useful organisms that can be employed tohydroxylate and/or glycosylate the compounds of the invention. Theorganisms can be mutants unable to produce the polyketide normallyproduced in that organism, the fermentation can be carried out on platesor in large fermentors, and the compounds produced can be chemicallyaltered after fermentation. Thus, Streptomyces venezuelae, whichproduces picromycin, contains enzymes that can transfer a desosaminylgroup to the C-5 hydroxyl and a hydroxyl group to the C-12 position. Inaddition, S. venezuelae contains a glucosylation acticity thatglucosylates the 2′-hydroxyl group of the desosamine sugar. This lattermodification reduces antibiotic activity, but the glucosyl residue isremoved by enzymatic action prior to release of the polyketide from thecell. Another organism, S. narbonensis, contains the same modificationenzymes as S. venezuelae, except the C-12 hydroxylase. Thus, the presentinvention includes the compounds produced by hydroxylation andglycosylation of the initially formed polyketides of the invention byaction of the enzymes endogenous to S. narbonensis and S. venezilelae.

[0071] Other organisms suitable for making compounds of the inventioninclude Micromonospora megalomicea, Streptomyces antibioticus, S.fradiae, and S. thermotolerans. S. antibioticus produces oleandomycinand contains enzymes that hydroxylate the C-6 and C-12 positions,glycosylate the C-3 hydroxyl with oleandrose and the C-5 hydroxyl withdesosamine, and form an epoxide at C-8-C-8a. S. fradiae contains enzymesthat glycosylate the C-5 hydroxyl with mycaminose and then the4′-hydroxyl of mycaminose with mycarose, forming a disaccharide. S.thermotolerans contains the same activities as S. fradiae, as well asacylation activities. Thus, the present invention provides the compoundsproduced by hydroxylation and glycosylation of the macrolide aglyconesof the invention by action of the enzymes endogenous to S. antibioticus,S. fradiae and S. thernotolerans. The modified polyketides of theinvention can also be produced in recombinant host cells that have beentransformed with genes that encode polyketide modification enzymes fromanother organism.

[0072] The present invention also provides methods and geneticconstructs for producing the glycosylated and/or hydroxylated compoundsof the invention directly in the host cell of interest. Thus, thepolyketides of the invention can be produced directly by feeding inSaccharopolyspora erythraea, Streptomyces antibioticits, Micromonosporamegalomicea, S. fradiae, and S. thennotolerans. A number of erythromycinhigh-producing strains of Saccharopolyspora erythraea have beendeveloped, and such strains can also be used to feed the diketidecompounds of the invention to produce modified polyketides.

[0073] Modification can also be effected by chemical means, such asglycosylation through cell-free preparations of appropriate glycosylasesor through chemical derivatization. Thus, a multiplicity of polyketidesand corresponding antibiotics may be obtained using the methods andcompounds of the invention.

[0074] In a specific embodiment of the invention, the diketide thioesterprepared from 4-pentenal is used to produce 15-ethenylerythromycins,which can be chemically converted into 15-(2-arylethyl)erythromycinanalogs such as 15-(2-(3-quinolyl)ethyl)erythromycin A and relatedcompounds:

[0075] These analogs are expected to provide an aromatic moiety suitablypositioned to interact with additional binding sites on the bacterialribosome, and thus exhibit enhanced antibacterial activity. Particularlypreferred analogs are the 6-O-methyl-3-descladinosyl-3-oxo analog andthe corresponding 11,12-cyclic carbamate (X=H,F):

[0076] Given the high structural similarity between the modularpolyketide synthases examined to date, it should be clear that theinvention will provide methods for production of novel polyketides usingmany different enzymes other than the erythromycin polyketide synthase.For example, the genes encoding the polyketide synthases for rapamycin,FK-506, soraphen, epothilone, rifamycin, picromycin, tylosin,spiramicin, niddamycin, and avermectin have been examined and found toshow high homologies.

[0077] The following examples are thus intended to illustrate, not tolimit, the invention.

Preparation A N,S-Diacyl Cysteamines

[0078] A. N,S-Diacetylcysteamine:

[0079] Cysteamine hydrochloride (50.0 g) is added to a 1-L 3-neck roundbottom flask fitted with a magnetic stir bar, 2 addition funnels, and apH electrode. Water (300 ml) is added and the stirred solution is cooledon ice. The pH is adjusted to 8.0 by addition of 8 N KOH. Aceticanhydride (125 ml) is placed in one addition funnel, and 8N KOH (350 ml)is placed in the other addition funnel. The acetic anhydride is addeddropwise to the cysteamine solution, with 8 N KOH being added so as tokeep the reaction pH at 8+/−1. After addition of acetic anhydride iscomplete, the pH was adjusted to 7.0 using 1 N HCl and the mixture isallowed to stir for 75 min on ice. Solid NaCl is added to saturation,and the solution is extracted 4 times using 400 ml portions of CH₂Cl₂.The organic extracts are combined, dried over MgSO₄, filtered, andconcentrated under reduced pressure to yield 68.9 g (97% yield) of apale yellow oil, which crystallizes upon standing at 4° C. ¹H-NMR(CDCl₃, 400 MHz): δ 6.43 (br s, 1H), 3.42 (q,2H,J=7), 3.03 (t,2H,J=7),2.36 (s,3H), 1.98 (s,3H). ¹³C-NMR (CDCl₃, 100 MHz): δ 196.09, 170.45,39.42, 30.56, 28.71, 23.06.

[0080] B. N,S-Dipropionylcysteamine:

[0081] A solution of cysteamine hydrochloride (100 g) in 750 mL of waterin a 2-L round bottom flask fitted with a 250 ml addition funnel and amagnetic stirrer was treated with potassium hydroxide (49.4 g). Sodiumbicarbonate (222 g) was added after complete dissolution of the KOH. Theaddition funnel was charged with propionic anhydride (237 mL), which wasadded to the reaction over a period of 1 hour. Upon completion ofaddition, the reaction was stirred vigorously for an additional 1 hour.Solid sodium chloride was added to saturation, and the solution wasextracted 4 times with 500 ml portions of CH₂Cl₂. The organic extractswere combined, dried over MgSO₄, filtered, and concentrated on rotaryevaporator to give 155.2 g (93% yield) of a pale yellow oil, whichcrystallizes upon standing at 4° C.; mp 48-49° C. ¹H-NMR (CDCl₃, 400MHz): δ 5.8 (br s, 1H); 3.44 (q,2H,J=6); 3.03 (t,2H,J=6); 2.59(q,2H,J=7); 2.19 (q,2H,J=7); 1.18 (t,3H,J=7); 1.14 (t,3H,J=7). ¹³C-NMR(CDCl₃, 100 MHz): δ 200.64, 174.05, 39.45 37.38, 29.52, 28.36, 9.74,9.60.

[0082] C. N,S-dibutyrylcysteamine:

[0083] Butyryl chloride (10.4 mL) was added dropwise to a solution ofcysteamine (3.86 g) and triethylamine (14 mL) in 150 mL ofdichloromethane at 0° C. After addition, the mixture is warmed toambient temperature and stirred for an additional hour. The mixture ispoured into water, and the organic phase is collected. The organics arewashed sequentially with water, 1N HCl, saturated NaHCO₃, and brine,then dried over MgSO₄, filtered, and evaporated to yield a colorlessoil. Crystallization yields a waxy solid. ¹H-NMR (CDCl₃): δ 6.0 (br s,1H), 3.44 (q,2H,J=6); 3.03 (t,2H,J=6); 2.55 (t,2H,J=7); 2.14 (t,2H,J=7);1.67 (m,4H); 0.98 (t,3H,J=7); 0.94 (t,3H,J=7). ¹³C-NMR (CDCl₃): δ200.00, 173.15, 45.86, 39.50, 38.52, 28.39, 19.09, 19.01, 13.66, 13.39.

[0084] D. N,S-dipentanoylcysteamine, N,S-dihexanoylcysteamine,N,S-diheptanoylcysteamine, and N,S-dioctanoylcysteamine:

[0085] These were prepared as in paragraph A by reaction of cysteaminehydrochloride with the appropriate anhydride or acid chloride.

Preparation B Preparation of N-Acylcysteamines

[0086] A. N-Acetylcysteamine:

[0087] N,S-diacetylcysteamine (42.64 g) is placed in a 2-L round bottomflask fitted with a magnetic stirrer, and dissolved in 1400 ml of water.The flask is purged with N₂, and the mixture is chilled on an ice bath.Potassium hydroxide (49.42 g) is added, and the mixture is stirred for 2h on ice under inert atmosphere. The pH is adjusted to 7 using 6 N HCl,and solid NaCl is added to saturation. The mixture is extracted 7 timeswith 500 ml portions of CH₂Cl₂. The organic extracts are combined, driedover MgSO₄, filtered, and concentrated under reduced pressure to yield30.2 g (96% yield) of product. This material is distilled immediatelyprior to use, bp 138-140° C./7 mmHg.

[0088] B. N-Propionylcysteamine:

[0089] A solution of N,S-dipropionylcysteamnine (18.9 g) in methanol(100 mL) is placed under a nitrogen atmosphere with stirring. A solutionof sodium methoxide (25 wt %) in methanol (ca. 22 mL) is added slowlyuntil analysis by thin-layer chromatography (1:1 ethyl acetate/hexane)reveals complete disappearance of starting material. Oxalic aciddihydrate (6.3 g) is added, then the mixture is vacuum filtered througha pad of Celite and evaporated to give a colorless oil. Purification bydistillation gives the product.

[0090] C. Additional N-acylcysteamines:

[0091] Using the procedure of paragraph A, the correspondingN-butyrylcysteamine, N-pentanoylcysteamine, hexanoylcysteamine,N-heptanoylcysteamine, and N-octanoylcysteamine were prepared.

EXAMPLE 1 Preparation of (2S,3R)-2-methyl-3-hydroxyhexanoateN-acetylcysteamine thioester

[0092] A.(4S)—N-[(2S,3R)-2-methyl-3-hydroxyhexanoyl]-4-benzyl-2-oxazolidinone

[0093] A dry, 2 L three-necked round bottomed flask equipped with a 500ml addition funnel, a low-temperature thermometer, and a stir bar wascharged with 19.84 g of N-propionyl-oxazolidinone, capped with septa andflushed with nitrogen. Anhydrous dichloromethane (100 ml) was added bycannula and the resulting solution was cooled to −65° C. in a bath ofdry ice/isopropanol. The addition funnel was charged by cannula with 100ml of dibutylboron triflate (1.0 M in dichloromethane), which was addedin a slow stream to the reaction. Triethylamine (15.6 ml) was addeddropwise by syringe, keeping the reaction temperature below −10° C. Thereaction was then transferred to an ice bath and allowed to stir at 0°C. for 30 minutes. After that period, the reaction was placed back intothe dry ice/isopropanol bath and allowed to cool to −65° C.Butyraldehyde (8.6 ml) was added rapidly by syringe, and the reactionwas allowed to stir for 30 min.

[0094] The reaction was transferred to an ice bath and the additionfunnel was charged with 100 ml of a 1 M aqueous phosphate solution, pH7.0 (the phosphate solution is comprised of equal molar amounts of mono-and dibasic potassium phosphate). The phosphate solution was added asquickly as possible while keeping the reaction temperature below 10° C.The addition funnel was then charged with 300 ml of methanol which wasadded as quickly as possible while keeping the reaction temperaturebelow 10° C. Finally, the addition funnel was charged with 300 ml of 2:1methanol-30% hydrogen peroxide. This was added dropwise to ensure thatthe temperature was kept below 10° C. The reaction was stirred for onehour after completion of addition. The solvent was then removed on arotary evaporator until a slurry remained. The slurry was extracted 4times with 500 ml portions of ethyl ether. The combined organic extractswere washed with 250 ml each of saturated aqueous sodium bicarbonate andbrine. The extract was then dried with MgSO₄, filtered, and concentratedto give a slightly yellow oil. The material was then chromatographed onSiO₂ using 2:1 hexanes:ethyl acetate (product Rf=0.4) resulting in 22.0g (85% yield) of title compound as a colorless oil. APCI-MS: m/z 306(MH+). ¹H-NMR (360 MHz, CDCl₃): δ 7.2-7.4 (5H,m, phenyl); 4.71(1H,m,H4); 4.17-4.25 (2H,m,H5); 3.96 (1H,m,H3′); 3.77 (1H,dq,J=2.5,7 Hz,H2′); 3.26 (1H,dd,J=4,13 Hz,benzylic); 2.79 (1H,dd,J=9,13 Hz,benzylic);1.5-1.6 (2H,m,H4′); 1.3-1.5 (2H,m,H5′); 1.27 (3H,d,J=7 Hz,2′-Me); 0.94(3H,t,J=7 Hz,H6′).

[0095] B. (2S,3R)-2-methyl-3-hydroxyhexanoate N-acetylcysteaminethioester

[0096] N-acetylcysteamine was distilled at 130° C./7 mmHg to give acolorless liquid at room temperature. A dry, 1 L three-necked roundbottomed flask equipped with a 500 ml addition funnel and a stir bar wascapped with septa and flushed with nitrogen. The flask was then chargedwith 10.7 ml of N-acetylcysteamine by syringe and with 400 ml ofanhydrous THF by cannula. The mixture was cooled with a MeOH/ice bath.Butyllithium (64 ml of 1.6 M in hexanes) was added dropwise by syringe,resulting in formation of a white precipitate. After stirring for 30min, trimethylaluminum (51 ml of 2.0 M in hexanes) was added dropwise bysyringe. The reaction became clear after addition of trimethylaluminumand was allowed to stir an additional 30 min. During this period, 20.5 g(0.068 mol) of(4S)—N-[(2S,3R)-2-methyl-3-hydroxylhexanoyl]-4-benzyl-2-oxazolidinonewas put under a blanket of nitrogen and dissolved in 100 ml of anhydrousTHF; this solution was then transferred in a slow stream by cannula intothe reaction. The resulting reaction mixture turned a yellow-green colorand was allowed to stir for 1 hr. The reaction was judged complete whenthe starting material could no longer be seen by thin-layerchromatographic analysis (ca. 1 hr).

[0097] The reaction was treated with enough saturated oxalic acid togive a neutral reaction with pH paper (approximately 90 ml). Thesolvents were then removed on a rotary evaporator to give a whiteslurry. The slurry was extracted six times with 250 ml portions of ethylether. The organic extracts were combined and washed with brine, driedwith MgSO₄, filtered, and concentrated to give a slightly yellow oil.The thioester product was purified by flash chromatography on SiO₂ using1:1 hexanes:EtOAc until the elution of 4-benzyl-2-oxazolidinone. At thatpoint, the solvent system was switched to 100% EtOAc to give purefractions of diketide thioester. The product fractions were combined andconcentrated to give 14.9 g (89% yield) of title compound. APCI-MS: m/z248 (MH+). ¹H-NMR (360 MHz, CDCl₃): δ 5.8 (br s,1H); 3.94 (dt,1H), 3.46(m,2H), 3.03 (dt,2H), 2.71 (dq,1H), 1.97 (s,3H), 1.50 (m,2H), 1.37(m,2H), 1.21 (d,3H), 0.94 (t,3H).

[0098] C. In a manner similar to that set forth in paragraph A, butsubstituting for N-acetylcysteamine, the various N-acylcysteaminesprepared in Preparation B, the corresponding N-acylcysteamine thioestersof (2S,3R)-2-methyl-3-hydroxyhexanoate were prepared.

EXAMPLE 2 Comparative Feeding of Diketide N-Acylcysteamine Thioesters

[0099] The N-acylcysteamine thioesters of(±)-(2S*,3R*)-2-methyl-3-hydroxy-hexanoate were fed to growing culturesof Streptomyces coelicolor CH999/pJRJ2, and the production of15-methyl-6-deoxyerythronolide B was monitored. Duplicate cultures weregrown in 50 ml of medium (sucrose (103 g/l), K₂SO₄ (0.25 g/l),MgCl₂.6H₂O (10.12 g/l), casaminoacids (0.1 g/l), yeast extract (5 g/l),TES buffer (5.73 g/l), sodium propionate (10 mM), and trace elements)supplemented with 50 ug/ml of thiostrepton. After 2 dayspost-inoculation, the cultures were fed with a solution of diketidethioester in 9:1 water/DMSO to give a final concentration of 0.5 mMdiketide thioester. Aliquots of the cultures were removed periodicallyand assayed for polyketide production by HPLC, with quantitationperformed by evaporative light scattering. Production of 15-methyl-6-dEB6 days after feeding was as follows: Yield of Acyl group 15-methyl-6-dEBAcetyl 26 mg/L Propionyl 35 mg/L Butyryl 30 mg/L Pentanoyl 35 mg/LHexanoyl 33 mg/L Heptanoyl 27 mg/L Octanoyl 23 mg/L

[0100] These preliminary results indicate that relatively littledifference in yield is obtained depending on the acyl group coupled tocysteamine, but that an optimum chain length at least with respect tothe diketide tested, is between 3-6C in the acyl group.

[0101] The following examples 3-6 describe the preparation of additionaloptically active forms of N-acylcysteamines

EXAMPLE 3 Preparation of (2S,3R)-2-methyl-3-hydroxy-4-pentenoateN-acetylcysteamine thioester

[0102] A.(4S)—N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinone

[0103] A dry, 2 L three-necked round bottomed flask equipped with a 500ml addition funnel, a low-temperature thermometer, and a stir bar wascharged with 20.0 g of propionyl oxazolidinone A, capped with septa andflushed with nitrogen. Anhydrous dichloromethane (100 ml) was added andthe resulting solution was cooled to −15° C. in a bath of methanol/ice.Dibutylboron triflate (100 ml of 1.0M in dichloromethane) was added in aslow stream via the addition funnel at such a rate as to keep thereaction temperature below 3° C. Diisopropylethylamine (17.9 ml) wasadded dropwise by syringe, again keeping the internal temperature below3° C. The reaction was then cooled to −65° C. using a dryice/isopropanol bath. Acrolein was added over 5 minutes by syringe. Thereaction was allowed to stir for 30 min after completion of addition.

[0104] The reaction was then transferred to an ice bath and the additionfunnel was charged with 120 ml (0.1 mol) of a 1 M aqueous phosphatesolution, pH 7.0 (the phosphate solution is comprised of equal molaramounts of mono- and dibasic phosphate). The phosphate solution wasadded as quickly as possible while keeping the reaction temperaturebelow 10° C. The addition funnel was then charged with 400 ml methanolwhich was added as quickly as possible while keeping the reactiontemperature below 10° C. Finally, the addition funnel was charged with400 ml of 2:1 methanol-30% hydrogen peroxide. This was added dropwise atfirst to ensure that the temperature was kept below 10° C. The reactionwas stirred for one hour. The solvent was then removed by rotaryevaporation until a slurry remained. The slurry was extracted 4 timeswith 500 ml portions of ethyl ether. The organic extracts were combinedand washed with 250 ml each of saturated sodium bicarbonate and brine,then dried with MgSO₄, filtered, and concentrated to give a slightlyyellow oil. Trituration with hexane induced crystallization.Recrystallization from ether by addition of hexane resulted in 13.67 g(55% yield) of product. ¹H-NMR (360 MHz, CDCl₃): δ 7.2-7.4 (m,5H); 5.86(ddd,1H), 5.35 (dt,1H), 5.22 (dt,1H), 4.71 (m,1H), 4.51 (m,1H), 4.21(m,2H), 3.89 (dq,1H), 3.26 (dd,1H), 2.80 (dd,1H), 1.25 (d,3H).

[0105] B. (2S,3R)-2-methyl-3-hydroxy-4-pentenoate N-acetylcysteaminethioester

[0106] N-acetylcysteamine was distilled at 130°/7 mm to give a colorlessliquid at room temperature. A dry, 1 L three-necked round bottomed flaskequipped with a 500 ml addition funnel and a stir bar was capped withsepta and flushed with nitrogen. The flask was then charged with 7.5 mlof N-acetylcysteamine by syringe and with 500 ml of anhydrous THF bycannula. The reaction was then cooled with a MeOH/ice bath. Butyllithium(44 ml of 1.6 M in hexane) was added dropwise by syringe. A whiteprecipitate formed as the n-BuLi was added. After stirring for 30 min,35.5 ml (0.071 mol) of trimethylaluminum (2.0 M in hexane) was addeddropwise by syringe. The reaction became clear after addition oftrimethylaluminum and was allowed to stir an additional 30 min:(4S)—N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinonefrom paragraph A (13.6 g) was put under a blanket of nitrogen, dissolvedin 50 ml of anhydrous THF, and this solution was then transferred in aslow stream by cannula into the reaction. The resulting reaction mixtureturned a yellow-green color and was allowed to stir for 1 hr. Thereaction was judged to be finished when starting material could nolonger be seen by thin-layer chromatography (ca. 30 min).

[0107] Enough saturated oxalic acid was added to give a neutral reactionwith pH paper (approximately 60 ml). The solvents were then removed byrotary evaporator to give a white slurry. The slurry was extracted sixtimes with 250 ml portions of ethyl ether The organic extracts werecombined, washed with brine, dried with MgSO₄, filtered, andconcentrated to give a slightly yellow oil. The thioester was thenpurified by flash chromatography on SiO₂. The column was run with 1:1hexanes:ethyl acetate until the elution of oxazolidinone. At that point,the eluent was switched to 100% ethyl acetate to give pure fractions ofproduct. The fractions were combined and concentrated to give 7.7 g (71%yield) of product. ¹H-NMR (360 MHz, CDCl₃): δ 5.82 (ddd,1H), 5.78 (br s,1H), 5.32 (dt,1H),5.21 (dt,1H), 4.47 (m,1H), 3.45 (m,2H), 3.04 (m,2H),2.81 (dq,1H), 1.96 (s,3H), 1.22 (d,3H).

EXAMPLE 4 Preparation of (2S,3R)-2-methyl-3-hydroxy-4-pentynoateN-acetylcysteamine thioester

[0108] A.(4S)—N-[(2S,3R)-2-methyl-3-hydroxy-5-trimethylsilyl-4-pentynoyl]-4-benzyl-2-oxazolidinone

[0109] Prepared according to the method of Example 1, paragraph A byreaction of (4S)—N-propionyl-4-benzyl-2-oxazolidinone with3-trimethylsilylpropargyl aldehyde in 80% yield.

[0110] B.(4S)—N-[(2S,3R)-2-methyl-3-hydroxy-4-pentynoyl]-4-benzyl-2-oxazolidinone

[0111] A solution of(4S)—N-[(2S,3R)-2-methyl-3-hydroxy-5-trimethylsilyl-4-pentynoyl]-4-benzyl-2-oxazolidinone(0.13 g) in 3 mL of dimethylformamide was treated with 48% aqueous HF(2.6 uL) and KF.2H₂O at ambient temperature for 100 min. Upon completionof the reaction, saturated aqueous sodium bicarbonate was added toneutralize the HF, and the mixture was extracted three times with equalportions of ether. The organic extracts were combined, filtered, anddried over MgSO₄. Filtration and evaporation gave the crude product,which was purified by silica gel chromatography (3:2 hexanes/ethylacetate) to yield 64 mg of product.

[0112] C. (2S,3R)-2-methyl-3-hydroxy-4-pentynoate N-acetylcysteaminethioester

[0113] In a 25 ml round bottom flask purged with N₂, N-acetyl cysteamine(0.12 ml, 1.1 mmol, 1.1 eq) was dissolved in 5.2 ml of anhydrous THF.The solution was cooled to 0° C. A 1.6 M solution of butyllithium inhexanes (0.68 ml, 1.1 mmol, 1.1 eq) was added with a syringe to give aheterogeneous mixture. A 2.0 M solution of trimethylaluminum in hexanes(0.55 ml, 1.1 mmol, 1.1 eq) was added dropwise with vigorous stirring togive a yellow-green solution. A solution of(4S)—N-[(2S,3R)-2-methyl-3-hydroxyl-4-pentynoyl]-4-benzyl-2-oxazolidinone (280 mg, 1.0 mmol, 1.0 eq) in 2ml of THF was added. The solution was stirred for 15 min and neutralizedwith saturated oxalic acid (aq). Volatiles were removed in vacuo. Theresulting slurry was extracted with 4×20 ml of ethyl acetate. Thecombined extracts were washed with a minimum of saturated aqueous CuSO₄to remove excess thiol. Some distilled water was used to aid separation.The organic layer was dried over MgSO₄, filtered and concentrated. Theresulting oil was purified by flash chromatography to give 191 mg oftitle compound (83% yield) as a pale yellow oil. ¹H-NMR (400 MHz,CDCl₃): δ 5.76 (br s,1H); 4.68 (dd,1H,J=2,4); 3.47 (m,2H); 3.05 (m,2H),2.9 (dq,1H), 2.8 (br d,1H); 2.51 (d,1H,J=2); 1.97 (s,3H); 1.38(d,3H,J=7).

[0114] D. Preparation of (2S,3R)-5-fluoro-2-methyl-3-hydroxypentanoateN-acetylcysteamine thioester

[0115] Prepared according to the procedure of Example 3, paragraph B,from(4S)—N-[(2S,3R)-5-fluoro-2-methyl-3-hydroxypentanoyl]-4-benzyl-2-oxazolidinoneand N-acetylcysteamine. ¹³C-NMR (100 MHz, CDCl₃): δ 203.53, 170.65,81.22 (d,J_(CF)=163), 68.48 (d,J_(CF)=4), 53.42, 39.12, 34.99(d,J_(CF)=26), 28.54, 23.07, 11.33.

EXAMPLE 5 Preparation of (4S,5R)-4-methyl-5-hydroxy-2-heptenoateN-acetylcysteamine thioester

[0116] A.(4S)—N-[(2S,4S,5R)-2,4-dimethyl-5-hydroxy-3-oxoheptanoyl]-4-benzyl-2-oxazolidinone

[0117] A solution of 2.0 g of(4S)—N-[(2S)-2-methyl-3-oxopentanoyl]-4-benzyl-2-oxazolidinone (preparedaccording to the procedure of Evans, et al., Tetrahedron (1992)48:2127-2142) in 18 ml of CH₂Cl₂ was cooled to −15° C., and 0.89 ml ofTiCl₄ was added dropwise over 3 minutes, followed by addition of 1.38 mlof diisopropylethylamine over 10 minutes. After stirring for 30 minutes,the mixture was cooled to −78° C. and 0.55 ml of propionaldehyde wasadded over 20 minutes. The mix was stirred overnight, then quenched with20 ml of saturated NH₄Cl and allowed to warm to ambient temperature.Water (5 ml) was added, and the resulting mixture was extracted threetimes with 75 ml portions of ether. The organic extracts were combined,washed with saturated NH₄Cl, saturated NaHCO₃, and brine, then driedover MgSO₄ and concentrated. The crude product was purified bychromatography on SiO₂ using a gradient from 9:1 to 1:1 hexanes/ethylacetate, yielding 1.9 gm (79%) of the product.

[0118] B.(4S)—N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl]-4-benzyl-2-oxazolidinone

[0119] Tetramethylammonium triacetoxyborohydride (2.89 g) was dissolvedin a mixture of acetic acid (11 ml) and acetonitrile (11 ml), stirredfor 30 minutes at ambient temperature, then cooled to −15° C. beforeaddition of(4S)—N-[(2S,4S,5R)-2,4-dimethyl-5-hydroxy-3-oxoheptanoyl]-4-benzyl-2-oxazolidinone(0.764 g). After stirring for 4 hours, 34 ml of 0.5 M sodium tartratewas added and stirring was continued for an additional 3 hours. Afterextraction with 3 portions of CH₂Cl₂, the organic phases were combinedand dried over MgSO₄. The solvent was removed under vacuum, and thecrude product was evaporated 3 times from 50 ml of methanol to yield0.644 g of product (84%).

[0120] C.(4S)—N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl]-4-benzyl-2-oxazolidinone3′,5′-cyclic carbonate

[0121] Triphosgene (0.138 g) was added to a −15° C. solution of(4S)—N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl]-4-benzyl-2-oxazolidinone(0.175 g), diusopropylethylamine (0.52 ml) and 4-dimethylaminopyridine(0.02 g) in 2 ml of CH₂Cl₂. After stirring for 16 hours, the reactionwas quenched by addition of 2 ml of sat. NH₄Cl and was extracted withethyl acetate. The organic extract was washed with sat. NH₄Cl and brine,then concentrated to give an orange oil. Chromatography (SiO₂) gave thepure cyclic carbonate (71 mg).

[0122] D.(4S)—N-[(4S,5R)-4-methyl-5-hydroxy-2-heptenoyl]-4-benzyl-2-oxazolidinone

[0123] A solution of(4S)—N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl]-4-benzyl-2-oxazolidinone3′,5′-cyclic carbonate (71 mg) in 4 ml of tetrahydrofuran was treatedwith 0.052 ml of diazabicycloundecene at ambient temperature for 16hours. Addition of 5 ml of sat. NH₄Cl followed by extraction with ethylacetate and evaporation of solvent yielded crude product, which waschromatographed on SiO₂ to give pure material (31 mg, 50%).

[0124] E. (4S,5R)-4-methyl-5-hydroxy-2-heptenoate N-acetylcysteaminethioester

[0125] A solution of N-acetylcysteamine (0.064 ml) in 3.4 ml oftetrahydrofuran at −15° C. was treated with 0.38 ml of 1.6 Mn-butyllithium in hexanes followed by 0.30 ml of 2.0 M trimethylaluminumin hexanes and stirred for 30 minutes. A 0.54 ml portion of thissolution was then added to a solution of 31 mg of(4S)—N-[(4S,5R)-4-methyl-5-hydroxy-2-heptenoyl]-4-benzyl-2-oxazolidinonein 0.3 ml of tetrahydrofuran and the mixture was stirred for 2 hoursbefore neutralization with saturated aqueous oxalic acid.

EXAMPLE 6 Additional Precursors to Optically Active N-Acyl CysteamineThioesters

[0126] A. Preparation of(4S)—N-[(2S,3R)-2-methyl-3-hydroxybutanoyl]-4-benzyl-2-oxazolidinone:

[0127] Prepared from (4S)-N-propionyl-4-benzyl-2-oxazolidinone andacetaldehyde according to the procedure described in Example 1,paragraph A. ¹H-NMR (360 MHz, CDCl₃): δ 7.2-7.4 (m,5H); 4.71 (m,1H);4.12-4.25 (m,2H); 3.76 (dq,1H); 3.26 (dd,1H); 2.79 (dd,1H); 1.30 (d,3H),1.21 (d,3H).

[0128] B. Preparation of(4S)—N-[(2S,3R)-2-vinyl-3-hydroxypentanoyl]-4-benzyl-2-oxazolidinone:

[0129] A solution of 2.45 g of (4S)-N-crotonyl-4-benzyl-2-oxazolidinonein 10 ml of anhydrous CH₂Cl₂ was cooled to −78° C., and 1.7 ml oftriethylamine was added followed by 10.5 ml of a 1 M solution ofdibutylboron triflate in CH₂Cl₂. After 30 minutes, the reaction waswarmed to 0° C., kept for 20 minutes, then recooled to −78° C.Propionaldehyde (0.9 ml) was added, and the reaction was allowed toslowly warm to ambient temperature over 16 hours. Standard oxidativeworkup yielded the product (1.98 g, 65% yield) after chromatography (2:1hexane/ethyl acetate). ¹H-NMR (360 MHz, CDCl₃): δ 7.2-7.35 (m,5H); 6.02(1H,m); 5.41 (m,2H); 4.72 (m,1H); 4.58 (dd,1H); 4.20 (m,2H); 3.92(m,1H); 3.25 (dd,1H); 2.98 (br s, 1H); 2.76 (dd,1H); 1.53 (m,2H); 0.98(t,3H).

[0130] C. Preparation of(4S)—N-[(2S,3R)-2-methyl-3-hydroxy-3-(3-pyridyl)propanoyl]-4-benzyl-2-oxazolidinone

[0131] Prepared from (4S)-N-propionyl-4-benzyl-2-oxazolidinone andpyridine-3-carboxaldehyde according to the procedure described inExample 1, paragraph A. ¹³C-NMR (100 MHz, CDCl₃): δ 176.40, 152.83,148.74, 147.85, 136.78, 134.80, 134.04, 129.34, 128.95, 127.45, 123.21,109.75, 71.46, 66.23, 55.05, 44.28, 37.70, 10.64.

[0132] D.(4S)—N-[(2S,3R)-5-fluoro-3-hydroxy-2-methylpentanoyl)]-4-benzyl-2-oxazolidinone

[0133] (a) A solution of 3-fluoropropanol in dichloromethane wasoxidized with the Dess-Martin periodinane. Analysis by ¹H-NMR revealedcomplete oxidation to 3-fluoropropanal. The suspension was filtered,washed with saturated sodium thiosulfate, then dried over MgSO₄. ¹H-NMR(CDCl₃, 400 MHz): δ 9.8 (t,1H), 4.8 (dt,2H), 2.85 (dt,2H).

[0134] (b) The aldol adduct was prepared according to the method ofExample 1, paragraph A by reaction of(4S)-N-propionyl-4-benzyl-2-oxazolidinone with the solution of3-fluoropropanal. ¹³C-NMR (100 MHz, CDCl₃): δ 177.08, 153.00, 134.94,129.37, 128.91, 127.39, 81.16 (d,J_(CF)=163), 67.67 (d,J_(CF)=4), 66.20,55.08, 42.23, 37.71, 34.52 (d,J_(CF)=19), 10.68.

[0135] Examples 7-14 illustrate the preparation of racemic diketidethioesters.

EXAMPLE 7 Preparation of 2-Benzoxazolone and ChlorozoxazoneIntermediates

[0136]

[0137] A. N-propionyl-2-benzoxazolone

[0138] A solution of 135 g of 2-benzoxazolone (1.0 mol) in 750 mL ofacetone was treated with 14 g (0.1 mol) of potassium carbonate and with130 mL (1.0 mol) of propionic anhydride at ambient temperature withstirring. After 4 hours, the mixture was poured into 3000 mL of waterwith vigorous stirring. The precipitated product was collected by vacuumfiltration, washed with water, and air dried to yield 187 g (98%) oflight tan-colored product suitable for further use; mp=88-90° C.(uncorr). Recrystallization from ether/hexane yields the pure product,172 g (90% yield), mp=92-93° C. (uncorr). ¹H-NMR (CDCl₃, 400 MHz): δ8.07 (1H,m); 7.21 (1H,m); 7.22-7.28 (2H,m); 3.12 (2H,q,J=7 Hz); 1.28(3H,t,J=7 Hz). ¹³C-NMR (CDCl₃, 100 MHz): δ 173.3, 151.3, 142.2, 127.8,125.1, 124.7, 115.9, 109.7, 30.4, 7.9.

[0139] B. N-propionylchlorzoxazone

[0140] A solution of 17 g of chlorzoxazone (5-chloro-2-benzoxazolone)(0.1 mol) in 75 mL of acetone was treated with 1.0 g (0.007 mol) ofpotassium carbonate and 15 mL (0.12 mol) of propionic anhydride atambient temperature with stirring. After 4 hours, the mixture was pouredinto 300 mL of water with vigorous stirring. The precipitated productwas collected by vacuum filtration, washed with water, and air dried toyield 22 g (98%) of colorless product; mp=97-99° C. (uncorr). ¹H-NMR(CDCl₃, 400 MHz): δ 8.11 (d, 1H, J=2 Hz); 7.23 (dd, 1H, J=2,9 Hz); 7.13(d, 1H, J=9 Hz); 3.12 (q, 2H, J=7 Hz); 1.28 (t, 3H, J=7 Hz). ¹³C-NMR(CDCl₃, 100 MHz): δ 173.08, 150.97, 140.67, 130.29, 128.45, 125.16,116.41, 110.59, 30.41, 7.89.

[0141] C. (±)—N-[(2R*,3S*)-(2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolone

[0142] A solution of N-propionyl-2-benzoxazolone (100.0 g) in anhydrousCH₂Cl₂ (1100 mL) was cooled to 3° C. with mechanical stirring under N₂atmosphere. TiCl₄ (58.4 mL) was added at a rate such that the internaltemperature remained below 10° C. (ca. 10 minutes). The resulting yellowslurry was stirred vigorously for 40 minutes, then triethylamine (87.4mL) was added at a rate such that the internal temperature remainedbelow 10° C. (ca. 10 minutes). The resulting deep red solution wasstirred for 80 minutes. Butyraldehyde (58.9 mL) was added at a rate suchthat the internal temperature remained below 10° C. (ca. 20 minutes),and the reaction was followed by thin-layer chromatography (4:1hexanes/ethyl acetate). After stirring for 90 minutes, the reaction wasquenched by addition of 450 mL of 2 N HCl. The phases were separated,and the aqueous phase was extracted 3 times with 750-mL portions ofether. The organic phases were combined and washed three times with200-mL portions of 2 N HCl. The acidic washes were combined andback-extracted 3 times with 150-mL portions of ether. The combinedorganic extract was washed once with 300 mL of sat. aq. NaHCO₃, and oncewith 300 mL of sat. aq. NaCl. The organic phase was then dried overMgSO₄, filtered, and concentrated under vacuum to a yellow slurry. Theproduct was collected by vacuum filtration and rinsed with hexanes toyield a colorless solid. Concentration of the filtrate yielded a secondcrop of product, which was collected in the same manner, giving acombined 103 g (80% yield) of crystalline product; mp=123-4° C. Themother liquor can be chromatographed (4:1 hexanes/ethyl acetate) toyield additional product. ¹H-NMR (CDCl₃, 400 MHz): δ 8.10 (1H,m),7.23-7.32 (3H,m), 4.12 (1H,m), 3.98 (1H,dq,J=3, 7), 2.26 (1H, br s),1.38-1.64 (4H,m), 1.34 (3H,d,J=7), 0.98 (3H,t,J=7). ¹³C-NMR (CDCl₃, 100MHz): δ 176.4, 151.1, 142.2, 127.8, 125.5, 124.9, 116.3, 109.9, 71.3,43.7, 36.2, 19.2, 13.9, 10.1.

[0143] D.(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxy-4-pentenoyl)]-2-benzoxazolone

[0144] This compound was prepared according to the procedure ofparagraph C, by reaction of N-propionyl-2-benzoxazolone with acrolein.¹H-NMR (CDCl₃, 400 MHz) δ 8.06 (m, 1 H), 7.27-7.20 (m, 2 H), 5.91 (ddd,J=17,10, 5 Hz). 5.37 (dt, J=1, 17 Hz, 1 H), 5.24 (dt, J=1, 10 Hz, 1 H),4.60 (m, 1 H), 4.06 (dq, J=3, 6 Hz, 1 H), 2.62 (d, J=4 Hz, 1 H), 1.30(d, 6 Hz, 3 H). ¹³C-NMR (CDCl₃, 100 MHz) δ 175.4, 151.1, 142.2, 137.2,127.7, 125.5, 124.9, 116.6, 116.2, 109.9, 72.7, 44.0, 10.7.

[0145] E.(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxyheptanoyl)]-2-benzoxazolone

[0146] This compound was prepared according to the method of paragraph Cby reaction of N-propionyl-2-benzoxazolinone with pentanal.

[0147] F.(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxy-6-heptenoyl)]-2-benzoxazolone

[0148] Was prepared according to the method of paragraph C by reactionof N-propionyl-2-benzoxazolinone with 4-pentenal. ¹³C-NMR (CDCl₃, 100MHz): δ 176.28, 151.07, 142.20, 137.96, 127.75, 125.50, 124.89, 116.26,115.19, 109.89, 70.93, 43.76, 33.12, 30.16, 10.21.

[0149] G. (±)—N-[(2R*,3S*)-(2-methyl-3-hydroxyoctanoyl)]-2-benzoxazolone

[0150] Was prepared according to the method of paragraph C by reactionof N-propionyl-2-benzoxazolinone with hexanal.

[0151] H.(±)—N-[(2R*,3S*)-(2,5-dimethyl-3-hydroxyhexanoyl)]-2-benzoxazolone

[0152] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolinone with 3-methylbutanal. ¹H-NMR (CDCl₃, 400MHz): δ 8.06 (m,1H); 7.25 (m,3H); 4.17 (m,1H); 3.91 (dq,1H,J=3,7); 2.52(br s,1H); 1.82 (m,1H); 1.56 (ddd,1H,J=5,9,13); 1.31 (d,3H,J=7); 1.25(ddd,1H,J=4,6,13); 0.95 (d,3H,J=7); 0.94 (d,3H,J=7).

[0153] I.(±)—N-[(2R*,3S*-(2-methyl-3-hydroxy-5-phenylentanoyl)]-2-benzoxazolone

[0154] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolinone with 3-phenylpropanal. ¹H-NMR (CDCl₃, 400MHz): δ 8.06 (m,1H); 7.25 (m,8H); 4.11 (dt,1H,J=4,7); 3.96(dq,1H,J=3,7); 2.91 (m,1H); 2.70 (m,1H); 1.95 (m,1H); 1.81 (m,1H); 1.34(t,3H,J=7).

[0155] J.(±)—N-[(2R*,3S*)-(4-(2-methoxyethoxy)-2-methyl-3-hydroxybutanoyl)]-2-benzoxazolone

[0156] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolinone with (2-methoxyethoxy)acetaldehyde.¹³C-NMR (CDCl₃, 100 MHz): δ 175.01, 151.02, 142.18, 127.91, 125.30,124.77, 116.15, 109.76, 73.23, 71.86, 70.73, 70.64, 58.88, 41.63, 11.88.

[0157] K.(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxy-3-phenylipropanoyl)]-2-benzoxazolone

[0158] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolinone with benzaldehyde; mp 155-158° C. ¹H NMR(CDCl₃): δ 8.10 (m, 1H), 7.45 (m, 2H), 7.35 (m, 2H), 7.26 (m, 4H), 5.30(d, 1H), 4.26 (dq, J=3, 6 Hz, 1 H), 1.26 (d, 3H). ¹³C NMR (CDCl₃): δ175.6, 151.0, 142.2, 141.0, 128.4, 127.7, 126.0, 125.5, 124.9, 116.3,110.0, 73.2, 46.0, 10.3.

[0159] L.(±)—N-[(2R*,3S*)-(5-azido-2-methyl-3-hydroxypentanoyl)]-2-benzoxazolone

[0160] (a) 3-azidopropanal was prepared by addition of HN₃ to acroleinaccording to A. J. Davies, et al. (1967) J. Chem. Soc., 2109-2112, andgave the following NMR data: ¹H-NMR (CDCl₃, 400 MHz): δ 9.80 (t, 1H, J=1Hz); 3.61 (t, 2H, J=7 Hz); 2.74 (dt, 2H, J=1,7 Hz). ¹³C-NMR (CDCl₃, 400MHz): δ 199.41, 44.42, 42.70.

[0161] (b) The aldol adduct of 3-azidopropanal andN-propionyl-2-benzoxazolone was prepared according to the procedure ofparagraph C. ¹H-NMR (CDCl₃, 400 MHz): δ 8.07 (m, 1H); 7.26 (m,3H); 4.23(dq, 1H, J=3,10 Hz); 3.96 (dq, 1H, J=3, 7 Hz); 3.52 (dd, 2H, J=6, 8 Hz);2.80 (dd, 1H, J=1,3 Hz); 1.84 (m, 2H); 1.75 (m, 2H); 1.34 (d, 3H, J=7Hz). ¹³C-NMR (CDCl₃, 100 MHz): δ 176.05, 151.07, 142.20, 127.63, 125.63,124.97, 116.26, 109.96, 68.85, 48.44, 43.80, 32.94, 10.48.

[0162] M.(±)—N-[(2R*,3S*)-(5-chloro-2-methyl-3-hydroxypentanoyl)]-2-benzoxazolone

[0163] (a) A solution of 3-chloropropanal in CH₂Cl₂ was prepared byaddition of HCl to acrolein according to the procedure described abovefor 3-bromopropanal, and gave the following NMR data: ¹H-NMR (CDCl₃, 400MHz): δ 9.78 (t, 1H, J=1 Hz); 3.80 (t, 2H, J=7 Hz); 2.93 (dt, 2H, J=1,7Hz). ¹³C-NMR (CDCl₃, 100 MHz): δ 198.77, 45.90, 36.79

[0164] (b) This solution was reacted with N-propionyl-2-benzoxazoloneaccording to the procedure of paragraph C to yield the product, whichwas crystallized from ether/hexane; mp=116-7° C. ¹³C-NMR (CDCl₃, 100MHz): δ 176.12, 151.06, 142.19, 127.63, 125.63, 124.96, 116.25, 109.96,68.40, 43.68, 41.70, 36.46, 10.55.

[0165] N.(±)—N-[(2R*,3S*)-(5-(2-pyrimidinylthio)-2-methyl-3-hydroxypentanoyl)]-2-benzoxazolone

[0166] (a) A suspension of 2-mercaptopyrimidine (6 g, 52 mmol) in ethylacetate (25 mL) was treated with 4 mL of acrolein and 100 mg oftetrabutylammonium hydroxide at 70° C. The bright yellow suspensionturned orange and cleared noticeably. After 30 min, the mixture wascooled, filtered, and evaporated to yield 8.53 gm (95%) of the productas an orange oil. ¹H-NMR (CDCl₃, 400 MHz): δ 9.83 (t, 1H, J=1 Hz); 8.51(d, 2H, J=5 Hz); 7.00 (t, 1H, J=5 Hz); 3.40 (t, 2H, J=7 Hz); 2.97 (dt,2H, J=1, 7 Hz). ¹³C-NMR (CDCl₃, 400 MHz): δ 200.51, 171.69, 157.30,116.66, 43.67, 23.31.

[0167] (b) The aldol adduct between 3-(2-pyrimidinyl-thio)propanal andN-propionyl-2-benzoxazolone was prepared according to the procedure ofparagraph C. ¹H-NMR (CDCl₃, 400 MHz): δ 8.49 (d, 2H, J=5 Hz); 8.07 (m,1H); 7.25 (m, 3H); 6.97 (t, 1H, 5 Hz); 4.25 (m, 1H); 4.01 (dq, 1H, J=3,7 Hz); 3.92 (br d, 1H, J=4 Hz); 3.32 (m, 2H); 2.05 (m, 1H); 1.95 (m,1H); 1.35 (d, 3H, J=7 Hz). ¹³C-NMR (CDCl₃, 100 MHz): δ 175.58, 172.66,157.28, 151.04, 142.16, 127.81, 125.38, 124.81, 116.51, 116.22, 109.81,69.62, 43.87, 34.29, 27.34, 10.89.

[0168] O.(±)—N-[(2R*,3S*)-(3-hydroxy-2,4,4-trimethylpentanoyl)]-2-benzoxazolone

[0169] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with trimethylacetaldehyde. Slow addition ofthe aldehyde to the enolate solution over 1 hour at 0° C. gave a 9:1ratio of the (2R*,3S*) and (2R*,3R*) isomers. The desired isomer wascrystallized from 1:1 ether/hexanes, mp=90-2° C. ¹H-NMR (CDCl₃, 400MHz): δ 8.04 (m, 1H), 7.23-7.29 (m, 3 H), 4.32 (dq,J=3, 7 Hz, 1H), 3.79(d,J=10 Hz, 1H), 3.41 (dd, J=3,10 Hz, 1 H), 1.51 (d,J=7 Hz, 3 H), 0.94(s, 9 H). ¹³C-NMR (CDCl₃, 100 MHz): δ 178.2, 150.8, 142.0, 127.6, 125.7,125.0, 116.3, 110.0, 84.0, 37.4, 36.2, 26.7, 18.1.

[0170] P.(±)—N-[(2R*,3R*)-(3-hydroxy-2,4,4-trimethylpentanoyl)]-2-benzoxazolone

[0171] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with trimethylacetaldehyde. Rapid additionof the aldehyde to the enolate solution at 0° C. gave a 1:1 ratio of the(2R*,3S*) and (2R*,3R*) isomers. The desired isomer was isolated bysilica gel chromatography, then crystallized. ¹-NMR (CDCl₃, 400 MHz): δ8.05 (m, 1 H), 7.20-7.27 (m, 4 H), 4.24 (dq, J=7, 4 Hz, 1 H), 3.82 (brt, J=4 Hz, 1 H), 2.33 (br d, J=4 Hz, 1 H), 1.36 (d, J=7 Hz, 3 H), 0.99,(s, 9 H). ¹³C-NMR (CDCl₃, 100 MHz): δ 175.3, 150.8, 142.2, 127.9, 125.4,124.9, 116.2, 109.9, 77.6, 40.3, 35.8, 29.7, 26.7, 12.7.

[0172] Q.(±)—N-[(2R*,3S*)-4-benzyloxy-3-hydroxy-2-methylbutanoyl)]-2-benzoxazoloneand(±)—N-[(2R*,3R*)-4-benzyloxy-3-hydroxy-2-methylbutanoyl)]-2-benzoxazolone

[0173] Prepared according to the procedure of paragraph C, by reactionof N-propionyl-2-benzoxazolone with benzyloxyacetaldehyde. The reactionyielded a 9:1 mixture of (2R*,3S*) and (2R*,3R*) isomers. The isomerswere separated by silica gel chromatography: (2R*,3S*): ¹H-NMR (CDCl₃,400 MHz): δ 8.03 (m, 1 H), 7.20-7.33 (m, 9 H), 4.55 (dd, J=25, 8 Hz, 2H), 4.26 (br q, J=5 Hz, 1 H), 4.10 (dq, J=5, 6 Hz, 1 H), 3.59 (m, 2 H),1.36 (d, 7 Hz, 3 H). ¹³C-NMR (CDCl₃, 100 MHz): δ 175.2, 151.0, 142.2,137.6, 128.4(2), 127.7(2), 125.4, 124.8, 116.2, 109.8, 7304, 71.6, 70.7,41.7, 11.9. (2R*,3R*): ¹H-NMR (CDCl₃, 400 MHz): δ 8.02 (m, 1 H),7.16-7.33 (m, 9 H), 4.52 (q, J=12 Hz, 2 H), 4.28 (p, J=7 Hz, 1 H), 4.04(br m, 1 H), 3.69 (dd, J=3, 10 Hz, 1 H), 3.64 (dd, J=5, 10 Hz, 1 H),3.09 (br s, 1 H), 1.31 (d, J=7H Hz, 3 H). ¹³C-NMR (CDCl₃, 100 MHz): δ175.8, 151.3, 142.0, 137.5, 128.3, 127.8, 127.7, 127.5, 125.3, 124.7,116.2, 109.7, 73.5, 73.4, 73.0, 41.1, 14.5.

[0174] R.(±)—N-[(2R*,3S*)-3-hydroxy-2-methyl-4-hexenoyl)]-2-benzoxazolone

[0175] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with trans-crotonaldehyde; mp 74-6° C. ¹HNMR (CDCl₃): δ 8.06 (m, 1 H); 7.23 (m, 3 H); 5.78 (dqd, 1 H, J=15, 7, 1Hz); 5.55 (ddq, 1 H, J=15, 7, 2 Hz); 4.52 (br, 1 H); 4.05 (qd, 1 H, J=7,4 Hz); 2.38 (br d, 1 H, J=3 Hz); 1.70 (ddd, 3 H, J=7, 1, 1 Hz, 3 H);1.30 (d, 3 H, J=7 Hz). ¹³C NMR (CDCl₃): δ 175.50, 151.20, 142.18,129.99, 128.78, 127.80, 125.43, 124.86, 116.22, 109.85, 72.92, 44.31,17.71, 11.04.

[0176] S. (±)-N-(2-(-1-hydroxycyclohexyl)propionyl)-2-benzoxazolone

[0177] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with cyclohexanone; mp 58-9° C. ¹H NMR(CDCl₃): δ 8.09 (m, 1 H); 7.25 (m, 3 H); 4.11 (q, 1 H, J=7 Hz); 2.90 (brs, 1 H); 1.82 (br d, 1 H, J=13 Hz); 1.59 (m, 8 H); 1.34 (d, 3 H, J=7Hz); 1.23 (m, 1 H). ¹³C NMR (CDCl₃): δ 177.20, 151.42, 142.03, 127.69,125.55, 124.91, 116.38, 109.88, 72.89, 46.77, 37.03, 33.37, 25.64,21.73, 21.40, 12.16.

[0178] T.(±)—N-[(2R*,3S*)-6-benzyloxy-3-hydroxy-2-methylhexanoyl)]-2-benzoxazolone

[0179] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 4-benzyloxybutyraldehyde.

[0180] U.(±)—N-[(2R*,3S*)-6,6,6-trifluoro-3-hydroxy-2-methylhexanoyl)]-2-benzoxazolone

[0181] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 4,4,4-trifluorobutyraldehyde. ¹H-NMR(CDCl₃, 400 MHz): δ 8.05 (m,1H); 7.25 (m,3H); 4.10 (dt,1H,J=3,10); 3.95(dq,1H,J=3,7); 2.65 (br s,1H); 2.43 (m,1H); 2.17 (m,1H); 1.76 (m,2H);1.33 (d,3H,J=7). ¹⁹F-NMR (CDCl₃, 386 MHz): δ −66.77.

[0182] V.(±)—N-[(2R*,3S*)-5-methylthio-3-hydroxy-2-methylpentanoyl)]-2-benzoxazolone

[0183] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 3-(methylthio)propionaldehyde. 1H-NMR(CDCl₃, 400 MHz): δ 8.06 (m,1H); 7.25 (m,3H); 4.25 (m,1H); 3.96(dq,1H,J=3,7); 2.82 (br d,1H); 2.68 (m,2H); 2.11 (s,3H); 1.90 (m,1H);1.78 (m,1H); 1.34 (d,3H,J=7).

[0184] W.(±)—N-[(2R*,3S*)-4-cyclohexyl-3-hydroxy-2-methylbutanoyl)]-2-benzoxazolone

[0185] Prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with cyclohexylacetaldehyde.

[0186] X.(±)—N-[(2R*,3S*)-5-(3-pyridyl)-3-hydroxy-2-methylpentanoyl)]-2-benzoxazolone

[0187] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 3-(3-pyridyl)propanal.

[0188] Y.(±)—N-[(2R*,3S*)-3-hydroxy-2-methyl-5-hexenoyl)]-2-benzoxazolone

[0189] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 3-butenal.

[0190] Z.(±)—N-[(2R*,3S*)-4-methoxy-3-hydroxy-2-methybutanoyl)]-2-benzoxazolone

[0191] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with methoxyacetaldehyde.

[0192] AA.(±)—N-[(2R*,3S*)-3-(2-methylthiazol-4-yl)-3-hydroxy-2-methylpropanoyl)]-2-benzoxazolone

[0193] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 2-methylthiazole-4-carboxaldehyde.

[0194] BB.(±)—N-[(2R*,3S*)-5-(2-methylthiazol-4-yl)-3-hydroxy-2-methylpentanoyl)]-2-benzoxazolone

[0195] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 3-(2-methylthiazol-4-yl)propanal.

[0196] CC.(±)—N-[(2R*,3S*)-3-hydroxy-2-methyl-5-heptynoyl)]-2-benzoxazolone

[0197] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with 3-pentynal.

[0198] DD.(±)—N-[(2R*,3S*)-3-(tetrahydrofuran-2-yl)-3-hydroxy-2-methylpropanoyl)]-2-benzoxazolone

[0199] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with tetrahydrofuran-2-carboxaldehyde.

[0200] EE.(±)—N-[(2R*,3S*)-5-(methoxycarbonyl)-3-hydroxy-2-methylpentanoyl)-]2-benzoxazolone

[0201] Is prepared according to the method of paragraph C by reaction ofN-propionyl-2-benzoxazolone with methyl 4-oxobutanoate.

[0202] FF.(±)—N-[(2R*,3S*)-5-fluoro-3-hydroxy-2-methylpentanoyl)]-2-benzoxazolone

[0203] (a) A solution of 3-fluoropropanol in dichloromethane wasoxidized with the Dess-Martin periodinane. Analysis by ¹H-NMR revealedcomplete oxidation to 3-fluoropropanal. The suspension was filtered,washed with saturated sodium thiosulfate, then dried over MgSO₄. ¹H-NMR(CDCl₃, 400 MHz): δ 9.8 (t,1H), 4.8 (dt,2H), 2.85 (dt,2H).

[0204] (b) The aldol adduct is prepared according to the method ofparagraph C by reaction of N-propionyl-2-benzoxazolone with the solutionof 3-fluoropropanal.

[0205] GG.(±)—N-[(2R*,3S*)-(5-phthalimido-2-methyl-3-hydroxypentanoyl)]-2-benzoxazolone

[0206] (a) 3-Phthalimidopropanal was prepared by addition of phthalimideto acrolein in the presence of tetrabutylammonium hydroxide according tothe procedure described by R. O. Atkinson & F. Poppelsdorf, J Chem. Soc.(1952) 2448. ¹H-NMR (CDCl₃, 400 MHz): δ 9.82 (t,1H,J=2); 7.85 (m,2H);7.72 (m,2H); 4.04 (t,2H,J=7); 2.88 (dt,2H,J=2,7). ¹³C-NMR (CDCl₃, 100MHz): δ 199.36, 167.98, 134.10, 131.95, 123.36, 42.35, 31.67.

[0207] (b) The aldol adduct was prepared by reacting3-phthalimidopropanal with N-propionyl-2-benzoxazolone according to theprocedure of paragraph C. ¹³C-NMR (CDCl₃, 100 MHz): δ 175.52, 168.75,151.02, 142.17, 134.02, 132.05, 127.75, 125.46, 124.87, 123.35, 116.29,109.85, 69.02, 44.00, 34.93, 33.11, 11.11.

[0208] HH.(±)—N-[(2R*,3S*)-(6-fluoro-2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolone

[0209] (a) 1-Bromo-3-fluoropropane is reacted with sodium cyanide togive 4-fluorobutyronitrile. The nitrile is reduced withdiisobutylaluminum hydride to give 4-fluorobutyraldehyde.

[0210] (b) The aldol adduct is prepared by reacting 4-butyraldehyde withN-propionyl-2-benzoxazolone according to the procedure of paragraph C.

EXAMPLE 8(±)—N-[(2R*,3S*)-(3-cyclopropyl-2-methyl-3-hydroxypropionyl)]chlorzoxazone

[0211]

[0212] A solution of N-propionylchlorzoxazone (2.25 g, 10 mmol) inanhydrous CH₂Cl₂ (50 mL) was cooled to 3° C. with mechanical stirringunder N₂ atmosphere. TiCl₄ (1.2 mL) was added at a rate such that theinternal temperature remained below 10° C. (ca. 1 minute). The resultingyellow slurry was stirred vigorously for 5 minutes, then triethylamine(1.5 mL) was added at a rate such that the internal temperature remainedbelow 10° C. (ca. 1 minutes). The resulting deep red solution wasstirred for 30 minutes. Cyclopropanecarboxaldehyde (0.75 mL) was addedin one portion. After stirring for 60 minutes, the reaction was quenchedby addition of 40 mL of 2 N HCl. The phases were separated, and theaqueous phase was extracted once with 40-mL of ether. The organic phaseswere combined, dried over MgSO₄, filtered, and concentrated under vacuumto a colorless oil. Recrystallization from 1:1 ether/hexanes yielded1.89 g of pure product. ¹H-NMR (CDCl₃, 400 MHz): δ 8.12 (d, 1H, J=2 Hz);7.25 (dd, 1 H, J=2, 8 Hz); 7.14 (d, 1H, J=8 Hz); 4.16 (dq, H, J=4, 7Hz); 3.28 (dd, 1H, J=4, 9 Hz); 2.22 (br s, 1H); 1.41 (d, 3H, J=7 Hz);1.10 (m, 1H); 0.56 (m, 2H); 0.37 (m, 2H). ¹³C-NMR (CDCl₃, 100 MHz): δ175.24, 150.7, 140.62, 130.42, 128.54, 125.40, 116.73, 110.64,44.45,14.93, 11.03, 3.47, 2.70.

EXAMPLE 9(±)—N-[(2R*,3S*)-(5-bromo-2-methyl-3-hydroxypentanoyl)]chlorzoxazone

[0213]

[0214] (a) A solution of 3-bromopropanal was prepared by bubblinganhydrous HBr into an ice-cold solution of acrolein (5.6 g, 100 mmol) indichloromethane (50 mL) containing 5 mg of dicinnamylacetone asindicator. Once the solution stayed red for 5 minutes after cessation ofHBr addition, the solution was checked by ¹H-NMR by addition of 20 uL to750 uL of CDCl₃. NMR revealed clean conversion to 3-bromopropanal, andrelative integration against the CH₂Cl₂ signal indicated a concentrationof 2.6 M 3-bromopropanal. Anhydrous MgSO₄ was added to the reactionmixture and stirred to absorb water. This solution was filtered and useddirectly in the subsequent aldol condensation. ¹H-NMR (CDCl₃, 400 MHz):δ 9.74 (t, 1H, J=1 Hz); 3.61 (t, 2H, J=7 Hz); 3.07 (dt, 2H, J=1,7 Hz).¹³C-NMR (CDCl₃, 400 MHz): δ 198.95, 45.96, 23.35.

[0215] (b) A solution of N-propionylchlorzoxazone (11.3 g, 50 mmol) inanhydrous CH₂Cl₂ (250 mL) was cooled to 3° C. with mechanical stirringunder N₂ atmosphere. TiCl₄ (6.0 mL) was added at a rate such that theinternal temperature remained below 10° C. (ca. 1 minute). The resultingyellow slurry was stirred vigorously for 30 minutes, then triethylamine(7.5 mL) was added at a rate such that the internal temperature remainedbelow 10° C. (ca. 1 minutes). The resulting deep red solution wasstirred for 30 minutes. The solution of 3-bromopropanal (25 mL, 60 mmol)was added in one portion. After stirring for 30 minutes, the reactionwas quenched by addition of 200 mL of 2 N HCl. The phases wereseparated, and the aqueous phase was extracted once with 200-mL ofether. The organic phases were combined and filtered through a pad ofsilica, washing the silica with ether. The filtrate was evaporated toyield a tan solid, which was recrystallized from ether by addition ofhexane to yield 9.5 g (52%) of the product as a colorless solid. ¹H-NMR(CDCl₃, 400 MHz): δ 8.14 (d, 1H, J=2 Hz); 7.28 (dd, 1 H, J=2, 8 Hz);7.18 (d, 1H, J=8 Hz); 4.33 (dt, 1H, J=3,10 Hz); 3.96 (dq, 1H, J=4, 7Hz); 3.61 (m, 2H); 2.3 (br s, 1H); 2.16 (m, 1H); 1.98 (m, 1H); 1.35 (d,3H, J=7 Hz). ¹³ C-NMR(CDCl₃, 100MHz): δ 175.76, 150.74, 140.62, 130.54,128.29, 125.64, 116.78, 110.78, 69.39, 43.71, 36.49, 30.14, 10.66.

EXAMPLE 10(±)—N-[(2R*,3S*)-(5-chloro-2-methyl-3-hydroxypentanoyl)]chlorzoxazone

[0216]

[0217] This was prepared according to the procedure for thecorresponding bromide of Example 9, using a solution of 3-chloropropanalin dichloromethane. ¹H-NMR (CDCl₃, 400 MHz): δ 8.12 (d, 1H, J=2 Hz);7.26 (dd, 1 H, J=2, 8 Hz); 7.17 (d, 1H, J=8 Hz); 4.33 (m, 1H); 3.94 (dq,1H, J=4, 7 Hz); 3.74 (m, 2H); 2.70 (br s, 1H); 2.05 (m, 1H); 1.90 (m,1H); 1.34 (d, 3H, J=7 Hz). ¹³C-NMR (CDCl₃, 100 MHz): δ 175.79, 150.74,140.62, 130.53, 128.29, 125.63, 116.77, 110.78, 68.40, 43.76, 41.64,36.37, 10.59.

EXAMPLE 11(±)—N-[(2R*,3S*)-3-hydroxy-2-vinyl-6-heptenoyl)]-2-benzoxazolone

[0218]

[0219] (a) N-crotonyl-2-benzoxazolone: A solution of 2-benzoxazolone(8.1 gm) in 60 mL of acetone was stirred with 8.3 gm of potassiumcarbonate while trans-crotonyl chloride (5.75 mL) was added dropwise.After 16 hours, the mixture was poured into 150 mL of water, and theresulting precipitate was collected by vacuum filtration and air dried.Recrystallization from ether/hexanes gave 10.5 gm (86%). ¹H-NMR (CDCl₃,400 MHz): δ 8.12 (m,1H); 7.40 (m,2H); 7.25 (m,4H); 2.07 (m,3H).

[0220] (b) Anhydrous CH₂Cl₂ (200 mL) was added to a flask containingN-crotonyl-2-benzoxazolone (8.00 g, 39.4 mmol, 1.00 eq) to make a 0.2 Msolution which was cooled to −78° C. in a dry ice/acetone bath. Titanium(IV) chloride (4.41 mL, 40.2 mmol, 1.02 equiv) was added dropwise. Theyellow slurry was stirred vigorously for 20 min. Freshly distilledtriethylamine (6.58 mL, 47.2 mmol, 1.20 equiv) was added dropwise. Thecolor changed from red-orange to deep purple during the addition. Thesolution was stirred for 1.5 h at −78° C. and 1.5 h at 0° C. Thereaction mixture was returned to −78° C.; and freshly distilled4-penten-1-al (bp 102-103° C.; 4.96 mL, 47.2 mmol, 1.2 equiv) was addeddropwise over 15 min. The solution was stirred for 2 h at −78° C. and1.5 h at 0° C. The color changed from purple to brown over this time.The reaction was quenched with 2 N HCl_((aq)) (1.5 eq). The mixture waspoured into a separatory funnel, and the layers were separated. Theorganic phase was vacuum filtered through a pad of silica. The silicawas washed with 3 volumes of ether, and all of the filtrate wasconcentrated. The crude material was chromatographed over silica (85:15hexanes:EtOAc) to give 8.05 g (71%) of a faintly colored oil. ¹³C-NMR(CDCl₃, 400 MHz): δ 173.25, 150.90, 142.13, 137.91, 130.42, 127.67,125.61, 124.91, 121.96, 116.19, 115.16, 109.92, 71.01, 53.79, 33.31,29.85.

EXAMPLE 12(4S)—N-[(1S,2R)-2-hydroxy-5-cyclohexenyl-1-carboxyl]-2-benzoxazolone

[0221]

[0222] A solution of(4S)—N-[(2S,3R)-3-hydroxy-2-vinyl-6-heptenoyl)]-4-benzyl-2-oxazolidinone(35 mg) and 8 mg of bis(tricyclohexylphosphine) benzylidenerutheniumdichloride (Grubbs' catalyst) in dichloromethane (5 mL) was heated atreflux under inert atmosphere for 1.5 h. Chromatography yielded thecyclic metathesized product. ¹³C-NMR(CDCl₃, 100MHz): δ 173.10, 153.56,135.09, 130.30, 19.42, 128.95, 127.39, 121.36, 67.10, 66.30, 55.34,45.57, 37.84, 27.44, 22.39.

EXAMPLE 13 Conversion to Thioesters

[0223] A. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoateN-propionylcysteamine thioester

[0224] One molar equivalent of sodium methoxide (25% w/v in methanol;ca. 150 mL) is added in a slow stream to a solution ofN,S-dipropionylcysteamine (173 g) in methanol (910 mL) under N₂. Whenhalf of the calculated volume has been added, the reaction is monitoredby TLC (1:1 ethyl acetate/hexanes), and methoxide addition is continueduntil complete conversion of the N,S-dispropionylcysteamine toN-propionylcysteamine.

[0225] The resulting solution of sodium N-propionylcysteamine thiolateis cannulated into a flask containing solid(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolone (240 g)under N₂. After 15 minutes, the reaction is quenched with solid oxalicacid dihydrate (80.4 g), filtered, and concentrated to a yellow oil. Theresidue is dissolved in 2:1 hexanes/ethyl acetate and submitted to batchelution chromatography on SiO₂. The silica is washed with 2:1hexanes/ethyl acetate to remove 2-benzoxazolone, then with ethylacetate/methanol (9:1) to elute the product thioester. Evaporation ofthe thioester-containing eluent yields 222 g of the thioester (98%yield) as a yellow oil, which crystallizes on standing; mp 37-39° C.¹H-NMR (CDCl₃, 400 MHz): δ 5.8 (br s, 1H); 3.93 (dt, 1H); 3.44 (m, 2H);3.03 (dt, 2H); 2.69 (dq, 1H); 2.19 (q, 2H); 1.47 (m, 2H); 1.36 (m, 2H);1.19 (d, 3H); 1.14 (t, 3H); 0.92 (t, 3H).

[0226] The following are prepared according to the method of paragraph Aof this example.

[0227] B. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate N-acetylcysteaminethioester:

[0228] Prepared according to the method of paragraph A, by reaction ofN,S-diacetylcysteamine and(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolone. ¹H-NMR(CDCl₃, 400 MHz): δ 6.1 (br s, 1H); 3.93 (dt, 1H); 3.44 (m, 2H); 3.03(dt, 2H); 2.72 (dq, 1H); 1.97 (s,3H); 1.51 (m, 2H); 1.37 (m, 2H); 1.23(d, 3H); 1.14 (t, 3H); 0.94 (t, 3H). ¹³C-NMR (CDCl₃, 100 MHz): δ 204.05,170.52, 71.87, 53.40, 39.33, 36.31, 28.53, 23.14, 19.16, 13.91, 11.14.

[0229] C. (±)-(2R*,3S*)-2-methyl-3-hydroxy-4-pentenoateN-propionylcysteamine thioester

[0230] Prepared according to the method of paragraph A, by reaction ofN,S-dipropionyl-cysteamine and(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxy-4-pentenoyl)]-2-benzoxazolone.

[0231] D. (±)-(2R*,3S*)-5-chloro-2-methyl-3-hydroxypentanoateN-acetylcysteamine thioester

[0232] Prepared according to the procedure of paragraph A fromN,S-diacetylcysteamine and(±)—N-[(2R*,3S*)-5-chloro-2-methyl-3-hydroxypentanoyl]chlorzoxazone.¹³C-NMR (CDCl₃, 100 MHz): δ 203.47, 170.76, 69.01, 53.43, 41.79, 39.13,36.80, 28.75, 23.17, 11.52.

[0233] E. (±)-(2R*,3S*)-5-bromo-2-methyl-3-hydroxypentanoateN-acetylcysteamine thioester

[0234] Prepared according to the procedure of paragraph A fromN,S-diacetylcysteamine and(±)—N-[(2R*,3S*)-5-bromo-2-methyl-3-hydroxypentanoyl]-chlorzoxazone.¹³C-NMR (CDCl₃, 100 MHz): δ 203.32, 170.82, 70.05, 53.44, 39.11, 37.00,30.41, 28.74, 23.18, 11.64.

[0235] F. (±)-(2R*,3S*)-5-azido-2-methyl-3-hydroxypentanoateN-acetylcysteamine thioester

[0236] Prepared according to the procedure of paragraph A fromN,S-diacetylcysteamine and(±)—N-[(2R*,3S*)-5-azido-2-methyl-3-hydroxypentanoyl]-2-benzoxazolone.¹³C-NMR (CDCl₃, 100 MHz): δ 203.41, 170.75, 69.43, 53.62, 48.46, 39.12,33.22, 28.73, 23.13, 11.57.

[0237] G. (±)-(2R*,3S*)-4-(2-methoxyethoxy)-2-methyl-3-hydroxybutanoateN-propionylcysteamine thioester

[0238] Prepared according to the procedure of paragraph A fromN,S-dipropionylcysteaamine and(±)-(±)—N-[(2R*,3S*)-(4-(2-methoxyethoxy)-2-methyl-3-hydroxybutanoyl)]-2-benzoxazolone.¹³C-NMR (CDCl₃, 100 MHz): δ 202.77, 174.05, 72.83, 71.89, 71.15, 70.66,58.97, 50.93, 39.30, 29.58, 28.61, 12.73, 9.72.

[0239] H.(±)-(2R*,3S*)-5-(2-pyrimidinylthio)-2-methyl-3-hydroxypentanoateN-propionylcysteamine thioester

[0240] Prepared according to the procedure of paragraph A fromN,S-dipropionylcysteamine and(±)-(±)—N-[(2R*,3S*)-(5-(2-pyrimidinylthio)-2-methyl-3-hydroxypentanoyl)]-2-benzoxazolone.¹³C-NMR (CDCl₃, 100 MHz): δ 203.23, 174.10, 157.30, 116.57, 70.12,53.62, 39.28, 34.56, 29.60, 28.61, 27.24, 14.17, 12.26, 9.74

[0241] I. (±)-(2R*,3S*)-2-methyl-3-hydroxy-6-heptenoateN-acetylcysteamine thioester

[0242] Prepared according to the procedure of paragraph A fromN,S-diacetylcysteamine and(±)—N-[(2R*,3S*)-(2-methyl-3-hydroxy-6-heptenoyl)]-2-benzoxazolone.¹H-NMR (CDCl₃, 400 MHz): δ 5.91 (br s,1H), 5.82 (m,1H), 5.06 (dq,1H),4.99 (dq,1H), 3.94 (m,1H), 3.47 (m,2h), 3.03 (m,2H), 2.73 (dq,1h), 2.56(br d,1H), 2.25 (m,1H), 2.15 (m,1H), 1.97 (s,3H), 1.60 (m,1H), 1.51(m,1H), 1.23 (d,3H). ¹³C-NMR (CDCl₃, 100 MHz): δ 203.96, 170.44, 137.93,115.18, 71.55, 53.40, 39.32, 33.30, 30.20, 28.60, 23.18, 11.26.

[0243] J. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate N-butyrylcysteaminethioester

[0244] Prepared according to the method of paragraph A usingN,S-dibutyrylcysteamine. ¹H-NMR (CDCl₃): δ 5.85 (br s,1H), 3.93 (m,1H),3.45 (m,2H), 3.02 (m,2H), 2.70 (dq,1H,J=3,7), 2.13 (m,3H), 1.65 (m,2H),1.49 (m,2H), 1.33 (m,2H), 1.21 (d,3H,J=7), 0.95 (t,3H,J=7), 0.92(t,3H,J=7).

[0245] K. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoateN-pentanoylcysteamine thioester

[0246] Prepared according to the method of paragraph A usingN,S-dipentanoylcysteamine. ¹H-NMR (CDCl₃): δ 5.81 (br s,1H), 3.92(m,1H); 3.44 (m,2H), 3.03 (m,2H), 2.70 (dq,1H,J=3,7), 2.15 (m,3H), 1.6(m,2H), 1.5 (m,2H), 1.35 (m,4H), 1.21 (d,3H,J=7), 0.93 (t,3H,J=7), 0.91(t,3H,J=7).

[0247] L. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate N-hexanoylcysteaminethioester

[0248] Prepared according to the method of paragraph A usingN,S-dihexanoylcysteamine. ¹H-NMR (CDCl₃): δ 5.83 (br s,1H), 3.92 (m,1H);3.44 (m,2H), 3.03 (m,2H), 2.69 (dq,1H,J=3,7), 2.14 (m,3H), 1.6 (m,2H),1.45 (m,2H), 1.30 (m,6H), 1.20 (d,3H,J=7), 0.93 (t,3H,J=7), 0.88(t,3H,J=7).

[0249] M. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoateN-heptanoylcysteamine thioester

[0250] Prepared according to the method of paragraph A usingN,S-diheptanoylcysteamine. ¹H-NMR (CDCl₃): δ 5.83 (br s,1H), 3.92(m,1H); 3.44 (m,2H), 3.03 (m,2H), 2.70 (dq,1H,J=3,7), 2.16 (m,3H), 1.6(m,2H), 1.49 (m,2H), 1.30 (m,8H), 1.20 (d,3H,J=7), 0.93 (t,3H,J=7), 0.87(t,3H,J=7).

[0251] N. (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate N-octanoylcysteaminethioester

[0252] Prepared according to the method of paragraph A usingN,S-dioctanoylcysteamine. ¹H-NMR (CDCl₃): δ 5.79 (br s,1H) 3.93 (m,1H);3.44 (m,2H), 3.03 (m,2H), 2.69 (dq,1H,J=3,7), 2.15 (m,3H), 1.6 (m,2H),1.49 (m,2H), 1.30 (m,10H), 1.21 (d,3H,J=7), 0.93 (t,3H,J=7), 0.87(t,3H,J=7).

EXAMPLE 14 (±)-(2S*,3R*)-2-vinyl-3-hydroxy-6-heptenoateN-pronionylcysteamine thioester

[0253]

[0254] N,S-Dipropionyl cysteamine (4.28 g, 22.6 mmol, 1.00 eq) wasdissolved in methanol (36 mL). A 25 wt % solution of sodium methoxide inmethanol (3.89 mL, 17.0 mmol, 0.750 eq) was added dropwise. The solutionwas stirred for 15 min and then cooled to −78° C. A methanolic solutionof the aldol adduct (6.50 g, 22.6 mmol, 1.00 eq in 9 mL of MeOH) wasadded dropwise. The reaction was stirred for 10 minutes at −78° C. andbrought up to room temperature before quenching with solid oxalic acid(1.42 g). Volatiles were removed in vacuo. The residue was redissolvedin ethyl acetate and washed with saturated NaHCO₃ followed by saturatedCuSO₄. The organic layer was dried over MgSO₄, filtered, concentrated,and chromatographed on silica gel (1:1 hexanes:EtOAc) to give 5.61 g(87.0%) of a colorless oil. ¹³C-NMR (CDCl₃, 100 MHz): δ 200.97, 174.15,137.88, 131.18, 121.63, 115.06, 71.02,64.45,38.97, 33.38, 29.83, 29.54,28.83, 9.68.

EXAMPLE 15 Production of 6-deoxyerythronolides

[0255] A. 15-methyl-6-deoxyerythronolide B

[0256] A seed culture of Streptomyces coelicolor K39-02/pJRJ2 was madeby inoculating 1 mL of frozen mycelium into a 2.8 L baffled flaskcontaining 500 mL of R2YE and shaking at 150-200 rpm/28-30° C. for 2days. A 10 L stirred tank bioreactor was prepared, filled with 10 L ofFKA medium, autoclaved at 121° C. for 30 min., allowed to cool, and theninoculated with 400-500 mL of seed culture.

[0257] Temperature was maintained at 28-30° C. with agitation providedby 3 rushton impellers at 500-750 rpm, aeration at ˜1 L/min., and pHcontrolled at 7.00 via automatic addition of 1 N NaOH or 1 N H₂SO₄.Glucose consumption, dissolved oxygen, pH, and cell mass were monitored.When the glucose concentration dropped below 0.1 g/L, the culture wassupplemented with 10 g of (±)-(2R*,3S*)-2-methyl-3-hydroxyhexanoateN-propionylcysteamine thioester in 50 mL of DMSO. Controlled feeding ofglucose maintained a glucose concentration of ˜0.5 g/L. Titers of15-methyl-6-deoxyerythronolide B were monitored by HPLC/MS, and theculture was harvested by centrifugation when the maximum titer wasreached.

[0258] The 15-methyl-6-deoxycrythronolide B was purified by solid phaseextraction. Fermentation broth was cooled to 4-15° C., and methanol wasadded to 10% (v/v). The broth was clarified by centrifugation and loadedonto an XAD-16 resin (Rohm and Haas) column (1 kg XAD/1 g15-methyl-6-deoxyerythronolide B) at a flow rate of 2-4 mL/cm²-min. Theloaded resin was washed with 2 column volumes of 15% (v/v) methanol inwater and the 15-methyl-6-deoxyerythronolide B was eluted from the resinwith acetone and collected in ½ column volume fractions. The fractionscontaining 15-methyl-6-deoxyerythronolide B were identified bythin-layer chromatography (ethyl acetate:hexanes 1:1) and HPLC/MS. Theacetone fractions containing 15-methyl-6-deoxyerythronolide B werepooled, and the volatiles were removed under reduced pressure. Theresulting aqueous mixture is extracted with ethyl acetate. The ethylacetate extract was washed with saturated NaH₂CO₃ and brine solutions,dried over sodium or magnesium sulfate, filtered, and concentrated todryness under reduced pressure. The crude material was purified bychromatography on silica gel using a gradient of hexanes and ethylacetate. Fractions containing the product were pooled and concentratedto a pale yellow oil that spontaneously crystallized. Recrystallizationfrom ether-hexane gave pure 15-methyl-6-deoxyerythronolide B. Massspectrometry shows [M+H]=401. ¹³C-NMR (CDCl₃, 100 MHz): δ 213.57,178.31, 79.51, 76.44, 74.47, 70.90, 43.95, 43.44, 40.88, 39.30, 37.66,37.48, 35.52, 34.37, 19.45, 16.58, 14.68, 13.73, 13.23, 9.22, 6.87,6.22.

[0259] B. 15-fluoro-6-deoxyerythronolide B

[0260] Prepared by feeding (2S,3R)-5-fluoro-3-hydroxy-2-methylpentanoateN-acetyl-cysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=405. ¹⁹F-NMR(CDCl₃, 376 MHz): δ −222.0 (relative to CF₃CO₂H at δ−77.0). ¹H-NMR(CDCl₃, 400 MHz): δ 5.49 (m,1H); 4.94 (m,2H); 3.99 (m,1H); 3.90(d,1H,J=10); 3.84 (d,1H,J=4); 3.70 (m,1H); 3.18 (br s,1H); 2.79 (m,1H);2.77 (m,1H); 2.61 (m,1H); 2.47 (br s,1H); 2.20 (m,1H); 2.00 (m,1H); 1.92(m,1H); 1.85 (m,1H); 1.70 (m,1H); 1.65 (dd,1H,J=4,10); 1.29 (d,3H,J=7);1.24 (dd,1H,J=4,10); 1.07 (d,3H,J=7); 1.06 (d,3H,J=7); 1.05 (d,3H,J=7);1.02 (d,3H,J=7); 0.93 (d,3H,J=7). ¹³C-NMR (CDCl₃, 100 MHz): δ 213.70,177.98, 80.68 (d,J_(CF)=167 Hz), 79.34, 76.37, 70.84 (d,J_(CF)=4 Hz),70.74, 43.88, 43.27, 41.13, 39.54, 37.63, 37.52, 35.52, 33.34(d,J_(CF)=20 Hz), 16.63, 14.60, 13.32, 9.20, 6.92, 6.28.

[0261] C. 14,15-dehydro-6-deoxyerythronolide B

[0262] Prepared by feeding (2S,3R)-3-hydroxy-2-methyl-4-pentenoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=385. ¹³C-NMR(CDCl₃, 100 MHz): δ 213.67, 177.51, 134.80, 116.58, 79.40, 76.47, 74.11,70.84, 43.80, 43.16, 41.48, 39.58, 37.61, 37.42, 35.56, 16.60, 14.55,13.34, 9.20, 6.91, 6.30.0

[0263] D. 15-chloro-6-deoxyerythronolide B

[0264] Prepared by feeding(±)-(2S*,3R*)-5-chloro-3-hydroxy-2-methylpentanoate N-acetylcysteaminethioester to S. coelicolor CH999/pJRJ2 according to the method ofparagraph A. The crude material is purified by silica gel chromatographyusing ethyl acetate/hexanes. APCI-MS: [M+H]=421.

[0265] E. 15-bromo-6-deoxyerythronolide B

[0266] Prepared by feeding(±)-(2S*,3R*)-5-bromo-3-hydroxy-2-methylpentanoate N-acetylcysteaminethioester to S. coelicolor CH999/pJRJ2 according to the method ofparagraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=465, 467.

[0267] F. 15-dimethyl-6-deoxyerythronolide B

[0268] Prepared by feeding (±)-(2S*,3R*)-2,5-dimethyl-3-hydroxyhexanoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=415. ¹³C-NMR(CDCl₃, 100 MHz): δ 213.98, 178.35, 79.58, 76.41, 72.87, 71.01, 43.96,43.48, 41.26, 41.16, 39.35, 37.65, 37.43, 35.43, 25.33, 22.99, 21.95,16.58, 14.56, 13.21, 9.29, 6.90, 6.24.

[0269] G. 15-phenyl-6-deoxverythronolide B

[0270] Prepared by feeding(±)-(2S*,3R*)-5-phenyl-3-hydroxy-2-methylpentanoate N-acetylcysteaminethioester to S. coelicolor CH999/pJRJ2 according to the method ofparagraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=463. ¹³C-NMR(CDCl₃, 100 MHz): δ 213.85, 178.30, 140.78, 128.55, 128.30, 126.23,79.45, 76.37, 74.19, 70.90,43.93, 43.37, 42.35, 41.04, 40.80, 39.47,37.56, 37.56, 35.47, 34.41, 32.58, 16.65, 14.80, 13.28, 9.28, 6.95,6.26.

[0271] H. 15-ethyl-6-deoxyerythronolide B

[0272] Prepared by feeding (±)-(2S*,3R*)-3-hydroxy-2-methylheptanoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=415. ¹³C-NMR(CDCl₃, 100 MHz): δ 213.65, 178.32, 79.51, 76.42, 74.74, 70.91, 43.95,43.44, 40.84, 39.32, 37.65, 37.48, 35.50, 31.96, 28.37, 22.32, 16.58,14.68, 13.93, 13.23, 9.20, 6.88, 6.23.

[0273] I. 15-propyl-6-deoxyerythronolide B

[0274] Prepared by feeding (±)-(2S*,3R*)-3-hydroxy-2-methyloctanoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=429. ¹³C-NMR(CDCl₃, 100 MHz): δ 213.66, 178.33, 79.51, 76.41, 74.76, 70.91, 43.95,43.44, 40.85, 39.31, 37.65, 37.47, 35.50, 32.23, 31.38, 25.86, 22.48,16.58, 14.68, 13.91, 13.22, 9.20, 6.88, 6.22.

[0275] J. 15-ethenyl-6-deoxyerythronolide B

[0276] Prepared by feeding (±)-(2S*,3R*)-3-hydroxy-2-methyl-6-heptenoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. The crude material was purified by silica gelchromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=413.

[0277] K. 13-desesthyl-13-phenyl-6-deoxyerythronolide B

[0278] Prepared by feeding(±)-(2S*,3R*)-3-phenyl-3-hydroxy-2-methylpropanoate N-acetylcysteaminethioester to S. coelicolor CH999/pJRJ2 according to the method ofparagraph A. The crude material is purified by silica gel chromatographyusing ethyl acetate/hexanes. APCI-MS: [M+H]=435.

[0279] L. 12-ethenyl-12-desmethyl-6-deoxyerythronolide B

[0280] Prepared by feeding (2S,3R)-3-hydroxy-2-vinylpentanoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. APCI-MS: [M+H]=398.

[0281] M. 12,15-bisethenyl-12-desmethyl-6-deoxyerythronolide B

[0282] Prepared by feeding (2S,3R)-3-hydroxy-2-vinyl-6-heptenoateN-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according tothe method of paragraph A. APCI-MS: [M+H]=425.

[0283] N. 15-azido-6-deoxyerythronolide B

[0284] Prepared according to the method of Example Y using(2S*,3R*)-5-azido-2-methylpentanoate N-acetylcysteamine thioester.APCI-MS: [MH+]=429.

EXAMPLE 16 Derivatives of dEB

[0285] A.12-desmethyl-13-desethyl-12,13-(cyclohexenyl)-6-deoxyerythronolide B

[0286] Prepared by treatment of12,15-bisethenyl-12-desmethyl-6-deoxyerythronolide B with Grubbs'catalyst according to the procedure of Example 12.

[0287] B. 15-bromo-14-hydroxy-6-deoxyerythronolide B

[0288] A solution of 14,15-dehydro-6-deoxyerythronolide B in aqueousacetonitrile is treated with N-bromosuccinimide. The mixture isevaporated to dryness, and the product is isolated by silica gelchromatography. ¹³C-NMR (CDCl₃, 100 MHz): δ 214.19, 175.82, 88.69,81.52, 78.96, 77.46, 76.22, 49.43, 46.69, 43.76, 43.30, 38.50, 35.60,34.30, 27.65, 18.06, 16.35, 15.53, 13.84, 13.06, 7.53.

EXAMPLE 17 Conversion of 6-deoxyerythronolides into erythromycins

[0289] A. Fermentations were conducted in 10 L (and 150 L) bioreactors.A 1 mL aliquot of frozen Sac. erythraea K40-67 mycelium was used toinoculate a seed culture in 500 mL of R2YE medium. The culture wasshaken at 150-200 rpm/28-30° C. in a 2.8 L baffled Fernbach flask for˜48 hr. A 10 L stirred tank bioreactor was prepared, filled with 10 L ofR2YE medium (70 L for the 150 L fermentation), autoclaved at 121° C. for45 min., allowed to cool, and then inoculated with 200 mL (1.4 L for the150 L fermentation) of seed culture. Temperature was maintained at28-30° C. with agitation provided by 2 rushton impellers at 500-700 rpm,aeration at 1 L/min., and pH controlled at 7.20 via automatic additionof 1 N NaOH or 1 N H₂SO₄. Foam was suppressed by addition of antifoam at1 mL/L. The pH was controlled to avoid potential product degradationinto enol ether and spiroketal. Sucrose consumption, glucose evolution,dissolved oxygen, pH, and absorbance at 600 nm (cell mass) weremonitored. After 24-36 hr., the culture was fed 300 mg (1.62 g for the150 L fermentation) of a 6-dEB derivative compound dissolved in 3 mL (15mL for the 150 L fermentation) of 100% ethanol. Fermentation continuedfor ˜68-85 additional hr., and the fermentation broth was harvested bycentrifugation. Titers of erythromycin A, B, C, and D analogs during thecourse of the fermentation were determined by electrospray MS analysis.

[0290] The erythromycins produced were purified by solid phaseextraction. Fermentation broth was brought to pH 8.0 by addition of NaOHand chilled to 4-15° C., and ethanol was added (0.1 L/L broth). Thebroth was clarified by centrifugation and loaded onto an XAD-16 resin(Rohm and Haas) column (1 kg XAD/1 g erythromycin derivative) at a flowrate of 2-4 mL/cm²-min. The loaded resin was washed with 2 columnvolumes of 15% (v/v) ethanol in water and the erythromycin derivativewas eluted from the resin with acetone and collected in ½ column volumefractions. The fractions containing the erythromycin derivative wereidentified by thin-layer chromatography and HPLC/MS.

[0291] The acetone fractions containing erythromycin analogs are pooledand the volatiles are removed under reduced pressure. The resultingaqueous mixture is extracted with ethyl acetate. The ethyl acetateextract is washed with saturated NaHCO₃ and brine solutions, dried oversodium or magnesium sulfate, filtered, and concentrated to dryness underreduced pressure. The crude material is dissolved in dichloromethane andloaded onto a pad of silica gel and washed with dichloromethane:methanol(96:4 v/v) until the eluent is no longer yellow. The desired material isthen eluted with dichloromethane:methanol:triethylamine (94:4:2 v/v) andcollected in fractions. Fractions containing erythromycin are identifiedby thin-layer chromatography, collected and concentrated under reducedpressure. This material is recrystallized from dichloromethane/hexanes.

[0292] B. 15-fluoroerythromycin A

[0293] Prepared by feeding 15-fluoro-6-deoxyerythronolide B to Sac.erythraea according to the method of paragraph A. The crude material waspurified by silica gel chromatography. APCI-MS: [M+H]=752. ¹H NMR (400MHz, CDCl₃): δ 5.27 (1H, dd, 10, 2 Hz); 4.87 (1H, d, 5); 4.53 (2H, dtd,40, 6, <1); 4.41 (1H, d, 7); 3.98 (3H, m); 3.86 (1H, d, 1); 3.56 (1H, d,7); 3.48 (1H, m); 3.31 (3H, s); 3.23, (1H, dd, 10, 7); 3.19 (1H, br s);3.08 (1H, qd, 7, 1); 3.00 (1H, br s, 8); 2.84 (1H, qd, 7.1); 2.70 (1H,m); 2.46 (1H, 7, 4); 2.36 (1H, d, 16), 2.30 (6H, s); 2.03-1.95 (2H, m);1.94 (1H, m); 1.73 (1H, br d, 15); 1.69 (1H, br d, 14); 1.57 (1H, m);1.47 (3H, s); 1.28 (3H, d, 6); 1.24 (3H, s); 1.22 (3H, d, 6); 1.21 (1H,ovrlp); 1.17 (3H, d, 7); 1.15 (3H, d, ovrlp); 1.14 (3H, s); 1.14 (3H, d,ovrlp); 1.10 (3H, d, 7). ¹³C NMR (100 MHz, CDCl₃): δ 222.0, 175.4,103.2, 96.3, 83.4, 82.3 (d, 170 Hz), 79.8, 77.9, 75.1, 74,3, 72.6, 72.5(d, 4 Hz), 70.9, 68.9, 68.5, 65.6, 65.6, 49.5, 45,2, 44.8, 40.3, 39.6,38.5, 37.7, 34.9, 29.5, 29.4 (d, 20 Hz), 28.7, 27.0, 21.5, 21.4, 18.6,18.2, 16.1 15,3, 11.9, 9.1.

[0294] C. 15-ethenylerythromycin A

[0295] Is prepared by feeding 15-ethenyl-6-deoxyerythronolide B to Sac.erythraea according to the method of paragraph A. The crude material ispurified by silica gel chromatography.

EXAMPLE 18 15-(2-(3-quinolyl)ethyl)erythromycin A

[0296]

[0297] (1) A solution of 15-ethenylerythromycin A (1 mmol) in 5 mL ofdichloromethane is treated with benzoic anhydride (1.5 mmol) andtriethylamine (1.5 mmol) at ambient temperature for 30 hours. Aqueous 5%Na₂CO₃ is added and stirred for 30 minutes, then the mixture isextracted with dichloromethane. The organic extracts are combined,washed with saturated aqueous NaHCO₃ followed by brine, dried overMgSO₄, filtered, and evaporated. Chromatography on silica gel providespure 2′-O-benzoyl-15-ethenylerythromycin A.

[0298] (2) A mixture of 2′-O-benzoyl-15-ethenylerythromycin A (1 mmol),palladium diacetate (0.2 mmol), tritolylphosphine (0.4 mmol), and3-bromoquinoline (2 mmol) in 8 mL of deoxygenated acetonitrile is cooledto −78° C., degassed, and sealed in a reaction tube. The mixture is keptat 50° C. with stirring for 30 hours, then cooled and opened and theacetonitrile removed under vacuum. The residue is dissolved in ethylacetate and washed successively with 5% aqueous Na₂CO₃, 2% aqueous Tris,and brine. After drying over Mg₂SO₄, the mixture is filtered andevaporated. Silica gel chromatography gives pure2′-O-benzoyl-15-(2-(3-quinolyl)ethyl)erythromycin A.

[0299] (3) A solution of2′-O-benzoyl-15-(2-(3-quinolyl)ethyl)erythromycin A (1 mmol) in methanol(10 mL) is heated at reflux for 6 hours, then evaporated. The residue ispurified by silica gel chromatography to yield15-(2-(3-quinolyl)ethyl)erythromycin A.

EXAMPLE 19 Preparation of Polystyrene-Supported 2-Benzimidazolone

[0300] (1) A mixture of 2-hydroxybenzimidazole,6-(acetylthio)-1-bromohexane, and triethylamine in acetonitrile isheated at reflux to prepare 1-(6-(acetylthio)hexyl)-2-benzimidazolone.

[0301] (2) A solution of 1-(6-(acetylthio)hexyl)-2-benzimidazolone inmethanol is treated with one equivalent of sodium methoxide to prepare1-(6-mercaptohexyl)-2-benzimidazolone.

[0302] (3) Merrifield resin (chloromethylatedpolystyrene-divinylbenzene) is suspended in dichloromethane by gentlestirring, and treated with 1-(6-mercaptohexyl)-2-benzimidazolone andtriethylamine to prepare polystyrene-supported 2-benzimidazolone.

EXAMPLE 20 Preparation of Polystyrene-Supported(4S)-4-benzyl-2-imidazolidinone

[0303] (1) N-ethoxycarbonyl-(L)-phenylalinal is prepared fromcommercially-available N-ethoxycarbonyl-(L)-phenylalanine according tothe method described for N-tbutoxycarbonyl-(L)-leucinal by O. P. Goel,et al., Organic Syntheses (1988) 67:69. This aldehyde is dissolved inmethanol and treated with 1,4-diaminobutane, acetic acid, and sodiumcyanoborohydride at 0° C. The resulting amine is isolated bychromatography, then heated under vacuum with removal of ethanol toprovide (4S)-1-(4-aminobutyl)-4-benzyl-2-imidazolinone.

[0304] (2) Carboxypolystyrene resin is suspended by gentle stirring indichloromethane and treated sequentially with 1-hydroxybenzotriazole anddicyclohexylcarbodiimide. After 30 minutes,(4S)-1-(4-aminobutyl)-4-benzyl-2-imidazolinone is added. The solution ischecked periodically for disappearance of the amine. The resin iscollected by vacuum filtration, washed with dichloromethane and dried.

EXAMPLE 21 General Solid-Phase Synthesis of(2S,3R)-2-Methyl-3-hydroxy-diketide thioesters

[0305] (1) Polystyrene-supported (4S)-4-benzyl-2-imidazolidinone issuspended in tetrahydrofuran and treated with excess propionicanhydride, triethylamine, and catalytic 4-dimethylaminopyridineovernight. The resin is collected by vacuum filtration and washed withwater followed by acetone, then dried under vacuum to yieldpropionylated resin.

[0306] (2) The propionylated resin is suspended by shaking in anhydrousdichloromethane in a bottom-fritted reaction vessel under inertatmosphere and cooled to 0° C. A small molar excess of dibutylborontriflate is added and the vessel contents are shaken for 30 minutes. Asmall molar excess of triethylamine is added and the vessel contents areshaken for another 30 minutes. The liquid phase is drained from thevessel through the bottom frit using gas pressure, and is replaced withclean dichloromethane containing a small molar excess of the aldehydecomponent. After shaking for 4 hours, the solvent is drained from thevessel via the frit and the resin is washed with clean dichloromethane.The resin is suspended in a mixture of phosphate buffer, pH 7, methanol,and H₂O₂ and shaken for 1 hour at 0° C. The solution is drained and theresin is washed sequentially with water, saturated NaHCO₃, water,methanol, and tetrahydrofuran, then dried under vacuum.

[0307] (3) An N-acylcysteamine is dissolved in tetrahydrofuran underinert atmosphere and cooled to −78° C. One molar equivalent ofn-butyllithium is added, resulting in a white suspension. Addition ofone molar equivalent of trimethylaluminum results in a clear solution ofthe aluminate salt. The resulting solution is added to thediketide-containing resin, and the mixture is shaken to release thediketide thioester. The solution is neutralized with oxalic acid,collected from the resin by vacuum filtration via the frit, andevaporated to dryness. The residue is resuspended in ethyl acetate andwashed with saturated aqueous CuSO₄ followed by brine. After drying overMgSO₄, the solution is filtered and evaporated. Chromatography yieldsthe purified diketide thioester.

EXAMPLE 22 Preparation of Polystyrene-Supported 2-Benzoxazolone

[0308] (1) A mixture of chlorzoxazone,3-(t-butoxycarbonylamino)-1-propene, palladium diacetate,tritolylphosphine, and acetonitrile is cooled to −78° C., degassed, andsealed in a reaction tube. The mixture is kept at 50° C. with stirringfor 60 hours, then cooled and opened and the acetonitrile removed undervacuum. The residue is dissolved in ethyl acetate and washedsuccessively with 5% aqueous NaHCO3 and brine. After drying over Mg2SO₄,the mixture is filtered and evaporated. Silica gel chromatography gives5-(3-(t-butoxycarbonylamino)-1-propenyl)-2-benzoxazolone.

[0309] (2) A solution of5-(3-(t-butoxycarbonylamino)-1-propenyl)-2-benzoxazolone intrifluoroacetic acid is stirred at ambient temperature for 30 min, thenevaporated to dryness to yield 5-(3-amino-1-propenyl)-2-benzoxazolone.

[0310] (3) Carboxypolystyrene resin is suspended by gentle stirring indichloromethane and treated sequentially with 1-hydroxybenzotriazole anddicyclohexylcarbodiimide. After 30 minutes,5-(3-amino-1-propenyl)-2-benzoxazolone is added. The solution is checkedperiodically for disappearance of the amine. The resin is collected byvacuum filtration, washed with dichloromethane and dried.

EXAMPLE 23 General Solid-Phase Synthesis of Racemic2-Methyl-3-hydroxy-diketide thioesters

[0311] (1) Polystyrene-supported 2-benzoxazolone is suspended in acetoneand treated with excess propionic anhydride and triethylamine overnight.The resin is collected by vacuum filtration and washed with waterfollowed by acetone, then dried under vacuum to yield propionylatedresin.

[0312] (2) The propionylated resin is suspended by shaking in anhydrousdichloromethane in a bottom-fritted reaction vessel under inertatmosphere and cooled to 0° C. A small molar excess of titaniumtetrachloride is added and the vessel contents are shaken for 30minutes. A small molar excess of triethylamine is added and the vesselcontents are shaken for another 30 minutes. The liquid phase is drainedfrom the vessel through the bottom frit using gas pressure, and isreplaced with clean dichloromethane containing a small molar excess ofthe aldehyde component. After shaking for 4 hours, the solvent isdrained from the vessel via the frit and the resin is washed with cleandichloromethane. The resin is washed with1 N HCl to remove titaniumresidues, followed by water and methanol. This provides2-methyl-3-hydroxy-diketides bound to polystyrene.

[0313] (3) An N,S-diacylcysteamine is dissolved in methanol and treatedwith one molar equivalent of methanolic sodium methoxide. The resultingsolution is added to the diketide-containing resin, and the mixture isshaken to release the diketide thioester. The solution is neutralizedwith oxalic acid, collected from the resin by vacuum filtration via thefrit, and evaporated to dryness. The residue is resuspended in ethylacetate and washed with saturated aqueous CuSO₄ followed by brine. Afterdrying over MgSO₄, the solution is filtered and evaporated.Chromatography yields the purified racemic diketide thioester.

EXAMPLE 2415-(2-(3-guinolyl)ethyl)-3-descladinosyl-3-oxo-6-O-methylerythromycin A11,12-cyclic carbamate

[0314]

[0315] A. 15-(2-(3-quinolyl)ethyl)erythromycin A-9-oxime

[0316] 15-(2-(3-quinolyl)ethyl)erythromycin A (25.7 g, 28.9 mmol, 1.00eq) is suspended in 42 mL of 2-propanol. Hydroxylamine (50 wt % in H₂O,22.2 mL, 375 mmol, 13.0 eq) is added. The mixture is stirred untilhomogeneous. Glacial HOAc is added. The solution is stirred at 50° C.for 11 h. Saturated NaHCO₃ is added. The mixture is concentrated andextracted with CHCl₃ (4×400 mL); washed with NaHCO₃ and water. Thecombined aqueous layers are back-extracted with 400 mL CHCl₃. Thecombined organic phases are washed with brine, dried over Na₂SO₄,filtered, and concentrated to yield the crude material. This is carriedon without further purification.

[0317] B. 15-(2-(3-quinolyl)ethyl)erythromycinA-9-(isopropoxycyclohexyl)oxime

[0318] The crude 15-(2-(3-quinolyl)ethyl)erythromycin A-9-oxime fromabove is dissolved in 72 mL of anhydrous CH₂Cl₂, and1,1-diisopropoxycyclohexane (29.2 mL, 140 mmol, 4.86 eq) is addeddropwise. A solution of pyridinium p-toluenesulfonate (10.5 g, 41.9mmol, 1.45 eq) in CH₂Cl₂ (36 mL) is added dropwise. Dichloromethane (200mL) is added after 15 h. The solution is washed with NaHCO₃ (2×100 mL)and water (100 mL). The combined aqueous phases are back-extracted with100 mL CH₂Cl₂. The combined organic layers are washed with brine, driedover MgSO₄, filtered, and concentrated. The material is chromatographedover silica gel to give the desired product.

[0319] C.2′,4″-Bis(O-trimethylsilyl)-15-(2-(3-quinolyl)ethyl)erythromycinA-9-(isopropoxycyclohexyl)oxime.

[0320] The 15-(2-(3-quinolyl)ethyl)erythromycinA-9-(isopropoxycyclohexyl)oxime (22.2 g, 21.3 mmol, 1.0 eq) is dissolvedin 54 mL anhydrous CH₂Cl₂ and cooled in an ice/water bath. A mixture ofchlorotrimethylsilane (4.05 mL, 31.9 mmol, 1.5 eq),N-(trimethylsilyl)-imidazole (7.81 mL, 53.2 mmol, 2.5 eq), and CH₂Cl₂(18 mL) is added dropwise. The reaction is stirred for 15 minutes aftercomplete addition and quenched with 600 mL EtOAc. The mixture is washedwith sat. NaHCO₃ (2×200 mL), water (200 mL), and brine (200 mL). Theorganic layer is dried over MgSO₄, filtered, and concentrated to yieldthe crude product which was carried on without further purification.

[0321] D.2′,4″-Bis(O-trimethylsilyl)-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA-9-(isopropoxycyclohexyl)oxime

[0322] Crude2′,4″-bis(O-trimethylsilyl)-15-(2-(3-quinolyl)ethyl)erythromycinA-9-(isopropoxycyclohexyl)oxime is dissolved in anhydroustetrahydrofuran (41 mL) and cooled to 10° C. Anhydrous methylsulfoxide(41.4 mL) and methyl bromide (2.0 M in ether, 20.7 mL, 41.4 mmol, 2.0eq) are added. A 1.0 M solution of potassium t-butoxide in THF (41.4 mL,41.4 mmol, 2.0 eq) is diluted with anhydrous methylsulfoxide (41.4 mL).This is added to the reaction mixture at a rate of 0.5 eq/hr. Thereaction is monitored by TLC (5:1 toluene:acetone). The reaction isquenched by the addition of ethyl acetate (200 mL) and sat. NaHCO₃ (70mL). The mixture is transferred to a separatory funnel and diluted with850 mL of ethyl acetate. The organic phase is washed with sat. NaHCO₃,water, and brine (300 mL each). The resulting emulsion is filteredthrough Celite. The separated organic phase is then dried over MgSO₄,filtered, and concentrated to give the crude product which is carried onwithout further purification.

[0323] E. 6-O-Methyl-15-(2-(3-quinolyl)ethyl)erythromycin A-9-oxime

[0324] The crude2′,4″-bis(trimethylsilyl)-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA-9-(isopropoxycyclohexyl)oxime from above is dissolved in acetonitrile(110 mL). Glacial acetic acid (67 mL) diluted with water (55 mL) isadded slowly. The solution is stirred 8 h. Toluene and 2-propanol areadded, and the solution is concentrated. The product is then dissolvedin toluene and concentrated twice to give the crude product which wascarried on without further purification.

[0325] F. 6-O-methyl-15-(2-(3-guinolyl)ethyl)erythromycin A

[0326] The crude 6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA-9-oxime from above and sodium hydrosulfite (23.1 g, 113 mmol, 5.63 eq)are placed in a round-bottom flask equipped with a condenser and flushedwith N₂. Ethanol (140 mL) and water (140 mL) are added. Formic acid(3.75 mL, 95.4 mmol, 4.77 eq) is added dropwise. The mixture is stirredat 80 C for 4.5 h. After the solution returned to room temperature, sat.NaHCO₃ was added. The pH is adjusted to 9-10 with 6 N NaOH. The mixtureis then extracted with 3×400 mL of ethyl acetate. The combined organicphases are washed with sat. NaHCO₃ then water (250 mL each). Thecombined aqueous phases are back-extracted with ethyl acetate (400 mL).The combined organic phases are washed with brine, dried over MgSO₄,filtered, and concentrated to give the crude product which was carriedon without further purification. Pure product can be obtained bychromatography on silica gel.

[0327] G.6-O-Methyl-15-(2-(3-guinolyl)ethyl)-3-descladinosylerythromycin A

[0328] The crude 6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycin A isstirred in 280 mL of 0.5 M HCl for 3 h. The pH is adjusted to 9-10 with6 N NaOH. The precipitate is collected by vacuum filtration and washedwith water. The mother liquor is extracted with 3×400 mL ethyl acetate.The combined organic phases are washed with sat. NaHCO₃ and water. Thecombined aqueous phases are back-extracted with ethyl acetate. Thecombined organic phases are washed with brine, dried over MgSO₄,filtered, and concentrated. The combined product is chromatographed oversilica gel the desired product as a white solid.

[0329] H.2′-O-Acetyl-6-O-methyl-15-(2-(3-quinolyl)ethyl)-3-descladinosylerythromycinA

[0330] 6-O-Methyl-15-(2-(3-quinolyl)ethyl)-3-descladinosyl erythromycinA (11.5 g, 15.5 mmol, 1.0 eq) is dissolved in 40 mL ethyl acetate. Asolution of acetic anhydride (2.92 mL, 31.0 mmol, 2.0 eq) in ethylacetate (35 mL) is added dropwise. The reaction is stirred for 30 minand then concentrated. The material is chromatographed over silica gelto give the desired product as a white solid.

[0331] I.2′-O-Acetyl-3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA

[0332] 2′-O-Acetyl-6-O-methyl-15-(2-(3-quinolyl)ethyl)-3-descladinosylerythromycin A (10 g, 12.8 mmol, 1.0 eq) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (16.51 g,86.1 mmol, 6.7 eq) are combined in a round-bottom flask and flushed withN₂. The solids are dissolved in anhydrous CH₂Cl₂ (64 mL) and cooled inan ice water bath. Anhydrous DMSO (15.5 mL, 218 mmol, 17 eq) is added. Asolution of pyridinium trifluoroacetate (12.14 g, 62.9 mmol, 4.9 eq) inCH₂Cl₂ (47 mL) is added over 3 h. The solution is diluted with 600 mL ofethyl acetate and washed with sat. NaHCO₃, water, and brine (200 mLeach). The organic phase is dried over MgSO₄, filtered, andconcentrated. Chromatography over silica gel gives the desired product.

[0333] J.2′-O-Acetyl-3-oxo-3-descladinosyl-11-methanesulfonyl-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA

[0334]2′-O-Acetyl-3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA is dissolved in freshly distilled pyridine (35 mL) and cooled in anice water bath. Methanesulfonyl chloride is added dropwise. The reactionis allowed to come to ambient temperature and stirred overnight. Ethylacetate (700 mL) is added, and the solution is washed with sat. NaHCO₃,water, and brine (200 mL each). The organic phase is dried over MgSO₄,filtered, and concentrated. Chromatography over silica gel gives thedesired compound.

[0335] K.2′-O-Acetyl-10,11-anhydro-3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA

[0336]2′-O-Acetyl-3-oxo-3-descladinosyl-11-methanesulfonyl-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA (6 g, 6.98 mmol, 1.0 eq) is dissolved in acetone (23 mL).1,8-Diazabicyclo(5.4.0)undec-7-ene (5.22 mL, 34.9 mmol, 5.0 eq) is addeddropwise. The reaction is stirred at ambient temperature for 4 h andthen concentrated. Chromatography over silica gel gave the desiredcompound.

[0337] L.3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycin A11,12-cyclic carbamate

[0338] A solution of2′-O-Acetyl-10,11-anhydro-3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycinA in dry tetrahydrofuran is added to a stirred suspension of NaH (3 eq.)in THF cooled to −10° C. To this is added a solution ofcarbonyldiimidazole (10 eq.) in THF/DMF (5:3), and the mixture isstirred for 2 hours. The reaction is warmed to ambient temperature anddiluted with concentrated aqueous ammonia and stirred overnight. Themixture is diluted with ethyl acetate and washed with aq. NaHCO₃ andbrine, dried over MgSO₄, and evaporated. Chromatography on silica gelyields the product.

[0339] The following provides additional products of the benzoxazolones.

[0340] A. Methyl (±)-(2S*,3R*)-3-hydroxy-2-methylhexanoate

[0341] 4-Dimethylaminopyridine (25 mg, 0.2 mmol) was added to a solutionof (±)—N-[(2S*,3R*)-(2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolone (263mg, 1.0 mmol) in methanol (10 mL). The reaction mixture was stirredovernight and the methanol was removed at reduced pressure. Theresulting oil was redissolved in ether (50 mL) and washed with 1 Nsodium hydroxide (2×10 mL), 2 N HCl (10 mL), and brine (10 mL), driedwith magnesium sulfate and concentrated at reduced pressure to give aclear oil (118 mg,74%). ¹H-NMR (CDCl₃, 400 MHz) δ 3.90 (m, 1 H),2.53(dq, J=3,3 Hz 1 H), 2.45 (br s, 1 H), 1.49 (m, 2 H), 1.34 (m, 2 H), 1.76(d, J=7 Hz), 0.93 (t, J=7 Hz). ¹³C-NMR (CDCl₃, 100 MHz) δ 176.3, 71.4,51.5, 44.3, 36.0, 19.0, 13.8, 10.6.

[0342] B. N-Benzyl (±)-(2S* 3R*)-3-hydroxy-2-methylhexanamide

[0343] Benzylamine (0.6 mL, 5.5 mmol) is added dropwise to a solution ofN-[(2S*,3R*)-3-hydroxy-2-methylhexanoyl]-2-benzoxazolinone (1.31 g, 5.0mmol) in 10 mL of tetrahydrofuran. A mildly exothermic reaction ensues.After 15 min, the solvent is evaporated. The residue is redissolved in50 mL of CH₂Cl₂ and washed successively with equal volumes of 1 N HCl, 1N NaOH, water, and brine. After drying over MgSO4, the solution isevaporated to yield 1.12 g (95% yield) of the product as a white solidwhich was recrystallized from ethyl acetate/hexanes as white needles, mp114-115° C. ¹H-NMR (d₆-DMSO, 400 MHz) δ 8.27 (t, J=6, 1 H), 7.30 (m, 2H), 7.22 (m, 3 H), 4.49 (d,J=6, 1 H), 4.27 (dd,J=6,15, 1 H), 4.20(dd,J=6,15, 1 H), 3.44 (m, 1 H), 2.20 (q, J=7, 1 H), 1.43 (m, 1 H), 1.22(m, 3 H), 1.04 (d,J=7,3 H), 0.79 (t,J=7,3 H). ¹³C-NMR (d₆-DMSO, 100MHz): 175.2, 140.2, 128.6, 127.6, 127.1, 71.8, 46.9, 42.2, 37.6, 19.0,14.7, 14.5.

1. A method to prepare a diketide or triketide thioester which methodcomprises a) treating a benzoxazolone derivative of said diketide ortriketide with the salt of a thiol anion from which the thioester is tobe formed so as to form the thioester of said diketide or triketide; orb) treating a 2-oxazolidinone derivative of said diketide or triketidewith the lithium salt of a thiol anion from which the thioester is to beformed in the presence of sufficient Lewis acid to reduce the basicityof the thiol anion so as to form the thioester of said diketide ortriketide.
 2. The method of claim 1 wherein said thioester is an N-acylcysteamine thioester.
 3. The method of claim 1 wherein the Lewis acid istrimethylammonium.
 4. A diketide or triketide thioester prepared by themethod of any of claims 1-3.
 5. A method to prepare a polyketide whichmethod comprises treating a polyketide synthase (PKS) enzyme complexwith the diketide or polyketide thioester of claim 4 under conditionswherein said polyketide is formed.
 6. The method of claim 5 wherein saidPKS is contained in a cell.
 7. The method of claim 5 wherein saidpolyketide has the lactone backbone structure of 6-dEB.
 8. A polyketideprepared by the method of any of claims 5-7.
 9. The polyketide of claim8 which contains a functional group in a side chain at position 13 ofthe lactone.
 10. The polyketide of claim 9 wherein the functional groupis a double bond, a triple bond, a halo group, an azide, an ester, analcohol, or an aromatic nucleus.
 11. The polyketide of claim 10 whereinthe functional group is a double bond, a halo group, an azide, or anaromatic nucleus.
 12. The polyketide of claim 8 which contains afunctional group at a side chain coupled to the 12 position of thelactone.
 13. The polyketide of claim 12 wherein said functional group isa double bond.
 14. A method to prepare a tailored polyketide whichmethod comprises treating the polyketide of claim 8 with tailoringenzymes.
 15. The method of claim 14 wherein the tailoring enzymes arecontained in a cell.
 16. A tailored polyketide prepared by the method ofclaim
 15. 17. The tailored polyketide of claim 16 which compriseshydroxylation or glycosylation.
 18. A method to prepare a derivatizedpolyketide or tailored polyketide which method comprises contacting thepolyketide of any of claims 9-13 or tailored polyketide of claim 16 or17 with a suitable reagent compatible with said functional group.
 19. Aderivatized polyketide or tailored polyketide prepared by the method ofclaim
 18. 20. The method of claim 1 wherein said diketide or triketidecomprises 2-methyl-3-hydroxy substituents.
 21. The method of claim 20wherein said diketide or triketide substituents have syn chirality. 22.The method of claim 21 wherein said diketide or triketide is selectedfrom the group consisting of 2-methyl-3-hydroxyhexanoyl;2-methyl-3-hydroxy-4-pentenoyl; 2-methyl-3-hydroxybutanoyl;2-vinyl-3-hydroxypentanoyl; and 2,4-dimethyl-3,5-dihydroxyheptanoyl. 23.The method of claim 1 wherein said diketide or triketide is selectedfrom the group consisting of (2S,3R)-2-methyl-3-hydroxyhexanoyl;(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl;(2S,3R)-2-methyl-3-hydroxybutanoyl; (2S,3R)-2-vinyl-3-hydroxypentanoyl;(2S,4S,5R)-2,4-dimethyl-5-hydroxy-3-oxoheptanoyl;(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl; and(4S,5R)-4-methyl-5-hydroxy-2-heptenoyl.
 24. A method to synthesize aderivative of at least a triketide containing stereochemically purechiral centers at at least positions 2 and 5 which method comprisestreating a stereochemically controlled diketide derivative having achiral center at position 2 of said diketide with an aldehyde in thepresence of tin(II) triflate and titanium tetrachloride so as tomaintain the chirality at position 2 and provide control of thechirality at position
 5. 25. The method of claim 24 wherein saidsterically controlled diketide is a derivative of 2-oxazolidinone.
 26. Amethod to synthesize an oligoketide thioester on a solid support, whichmethod comprises (1) reacting an N-acyl-2-imidazolidinone coupled tosaid solid support with an aldehyde or acyl moiety under conditionswhereby said aldehyde or acyl moiety couples to a position α to acarbonyl in the acyl group of the 2-imidazolidinone; (2) optionallyrepeating step (1); and (3) cleaving the resulting oligoketide from thesolid support by reaction with a salt of a thiol anion, thus providingan oligoketide thioester.
 27. The method of claim 26 wherein the salt isa lithium salt, and/or wherein said cleaving is performed in thepresence of a Lewis acid.
 28. A method to synthesize an oligoketidethioester on a solid support, which method comprises (1) reacting anN-acyl benzoxazolone coupled to said solid support with an aldehydeunder conditions whereby said aldehyde couples to a position α to acarbonyl in the acyl group of the benzoxalozone; (2) optionallyrepeating step (1); and (3) cleaving the resulting oligoketide from thesolid support by reaction with a salt of a thiol anion, thus providingan oligoketide thioester.
 29. A method to synthesize a racemic mixtureof diketides which method comprises treating an N-acyl derivative of abenzoxazolone with an aldehyde under conditions wherein said aldehydecouples to a position alpha to the carbonyl in the acyl group therebyobtaining a racemic mixture of diketides coupled to said benzoxazolone.30. The tailored polyketides of claim 16 selected from the groupconsisting of those of Examples 17B and 17C.
 31. The derivatizedpolyketide or tailored polyketide of claim 19 which is selected from thegroup consisting of the polyketides of Examples 16A, 16B, 18 and 24.